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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/16—Primer sets for multiplex assays

Definitions

• the present invention is as defined in the appended claims.
• the invention relates to methods of detecting a genetic variation in a genetic sample from a subject.
• the invention further relates to methods of detecting a genetic variation in a genetic sample from a subject using labeled probes and counting the number of labels in the probes.
• the invention relates to methods of detecting a genetic variation in a genetic sample from a subject.
• the genetic variation herein may include, but is not limited to, one or more substitution, inversion, insertion, deletion, or mutation in nucleotide sequences (e.g., DNA and RNA) and proteins (e.g., peptide and protein), one or more rare allele, polymorphism, single nucleotide polymorphism (SNP), large-scale genetic polymorphism, such as inversions and translocations, differences in the abundance and/or copy number (e.g., copy number variants, CNVs) of one or more nucleotide molecules (e.g., DNA), trisomy, monosomy, and genomic rearrangements.
• nucleotide sequences e.g., DNA and RNA
• proteins e.g., peptide and protein
• SNP single nucleotide polymorphism
• large-scale genetic polymorphism such as inversions and translocations
• the genetic variation may be trisomy 13, trisomy 18, trisomy 21, aneuploidy of X (e.g., trisomy XXX and trisomy XXY), or aneuploidy of Y (e.g., trisomy XYY).
• the genetic variation may be in region 22q11.2, 1q21.1, 9q34, 1p36, 4p, 5p, 7q11.23, 11q24.1, 17p, 11p15, 18q, or 22q13.
• the genetic variation may be a microdeletion or microamplification.
• the genetic sample may be a fetal genetic material from a maternal blood sample.
• the fetal genetic material may be isolated and separated from the maternal blood sample.
• the genetic sample is a mixture of fetal and maternal genetic material.
• the genetic sample may include aberrant genetic sequences arising from tumor formation or metastasis, and/or donor DNA signatures present in a transplant recipient
• the method may comprise isolating the plasma from a blood sample of the subject.
• genetic sample is serum
• the method may comprise isolating the serum from a blood sample of the subject.
• the genetic sample is a cell free DNA (cfDNA) sample
• the method further comprises isolating the cell free DNA sample from a sample obtained from the source described herein.
• the cell free DNA sample herein means a population of DNA molecules circulating freely in the bloodstream, outside of any cell or organelle. In the case of a pregnancy, cell free DNA from the mother carries a mixture of both maternal DNA as well as fetal DNA. These examples are not to be construed as limiting the sample types applicable to the present invention.
• Reference sequence used herein denotes a sequence to which a locus of interest in a nucleic acid is being compared. In certain embodiments, a reference sequence is considered a "wild type" sequence for a locus of interest.
• a nucleic acid that contains a locus of interest having a sequence that varies from a reference sequence for the locus of interest is sometimes referred to as "polymorphic” or “mutant” or “genetic variation.”
• a nucleic acid that contains a locus of interest having a sequence that does not vary from a reference sequence for the locus of interest is sometimes referred to as "non-polymorphic" or "wild type” or “non-genetic variation.”
• a locus of interest may have more than one distinct reference sequence associated with it (e.g., where a locus of interest is known to have a polymorphism that is to be considered a normal or wild type).
• the method may also comprise electing and/or isolating peptide or peptides
• the region of interest described herein may include "consensus genetic variant sequence” which refers to the nucleic acid or protein sequence, the nucleic or amino acids of which are known to occur with high frequency in a population of individuals who carry the gene which codes for a protein not functioning normally, or in which the nucleic acid itself does not function normally.
• the region of interest described herein may include "consensus normal gene sequence” which refers to a nucleic acid sequence, the nucleic acid of which are known to occur at their respective positions with high frequency in a population of individuals who carry the gene which codes for a protein not functioning normally, or which itself does not function normally.
• control region that is not the region of interest or the reference sequence described herein may include "consensus normal sequence" which refers to the nucleic acid or protein sequence, the nucleic or amino acids of which are known to occur with high frequency in a population of individuals who carry the gene which codes for a normally functioning protein, or in which the nucleic acid itself has normal function.
• the methods described herein may produce highly accurate measurements of genetic variation.
• One type of variation described herein includes the relative abundance of two or more distinct genomic loci.
• the loci may be small (e.g., as small as about 300, 250, 200, 150, 100, or 50 nucleotides or less), moderate in size (e.g., from 1,000, 10,000, 100,000 or one million nucleotides), and as large as a portion of a chromosome arm or the entire chromosome or sets of chromosomes.
• the results of this method may determine the abundance of one locus to another.
• the precision and accuracy of the methods of the present disclosure may enable the detection of very small changes in copy number (as low as about 25, 10, 5, 4, 3, 2, 1, 0.5, 0.1,0.05, 0.02 or 0.01 % or less), which enables identification of a very dilute signature of genetic variation.
• a signature of fetal aneuploidy may be found in a maternal blood sample where the fetal genetic aberration is diluted by the maternal blood, and an observable copy number of change of about 2% is indicative of fetal trisomy.
• the subject is a pregnant subject
• the genetic variation is a variation in the fetus of the pregnant subject in a region selected from the group consisting of 22q11.2, 1q21.1, 9q34, 1p36, 4p, 5p, 7q11.23, 11q24.1, 17p, 11p15, 18q, and 22q13, (e.g., a mutation and/or copy number change in any of regions 22q11.2, 1q21.1, 9q34, 1p36, 4p, 5p, 7q11.23, 11q24.1, 17p, 11p15, 18q, and 22q13).
• Fetus described herein means an unborn offspring of a human or other animal.
• the probe product, ligated probe set, conjugated probe set, ligated probes, conjugated probes, and labeled molecules may be single, contiguous molecule resulting from the performance of enzymatic action on a probe set, such as an assay.
• a probe product or a labeled molecule one or more individual probes from a probe set may be covalently modified such that they form a singular distinct molecular species as compared to either probes or probe sets.
• probe products or a labeled molecule may be chemically distinct and may therefore be identified, counted, isolated, or further manipulated apart from probes or probe sets.
• Contacting the probe sets to the genetic sample may be performed simultaneously or after hybridizing, ligating, amplifying and/or immobilizing the probes. Moreover, contacting the probe sets to the genetic sample may be performed simultaneously or before hybridizing, ligating, amplifying, and/or immobilizing the probes.
• a probe set comprises at least one probe that hybridize, conjugate, bind, or immobilize to a target molecule, including nucleic acids (e.g., DNA and RNA), peptides, and proteins.
• a probe may comprise an isolated, purified, naturally-occurring, non-naturally occurring, and/or artificial material, for example, including oligonucleotides of any length (e.g., 5, 10, 20, 30, 40, 50, 100, or 150 nucleotides or less), in which at least a portion(s) (e.g., 50, 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) of the oligonucleotide sequences is complementary to a sequence motif and/or hybridization domain present in one or more target molecules, such that the probe is configured to hybridize (or interact in a similar manner) in part or in total to one or more target molecules or nucleic acid region of interest.
• a probe may be single-stranded or double-stranded. Sometimes, the probe may be prepared from a purified restriction digest or produced synthetically, recombinantly or by PCR amplification. Sometimes, the probe may comprise a material that binds to a particular peptide sequence.
• a probe set may comprise a set of one or more probes designed to correspond to a single genomic location or a peptide in a protein sequence.
• Nucleotide used herein means either a deoxyribonucleotide or a ribonucleotide or any nucleotide analogue (e.g., DNA and RNA).
• Nucleotide analogues include nucleotides having modifications in the chemical structure of the base, sugar and/or phosphate, including, but not limited to, 5'- position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, substitution of 5-bromo-uracil, and the like; and 2'-position sugar modifications, including but not limited to, sugar-modified ribonucleotides in which the 2'- OH is replaced by a group selected from H, OR, R, halo, SH, SR, NH 2 , NHR, NR 2 , or CN.
• shRNAs also may comprise non-natural elements such as non-natural nucleotides, e.g., ionosin and xanthine, non-natural sugars, e.g., 2'-methoxy ribose, or non-natural phosphodiester linkages, e.g., methylphosphonates, phosphorothioates and peptides
• the shRNA may further comprise an element or a modification that renders the shRNA resistant to nuclease digestion.
• Polynucleotide or "oligonucleotide” is used interchangeably and each means a linear polymer of nucleotide monomers.
• Monomers making up polynucleotides and oligonucleotides are capable of specifically binding to a natural and/or artificial polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, base stacking, Hoogsteen or reverse Hoogsteen types of base pairing, or the like.
• Such monomers and their internucleosidic linkages may be naturally occurring or may be analogues thereof, e.g., naturally occurring or non-naturally occurring analogues.
• Polynucleotides typically range in size from a few monomeric units when they are referred to as “oligonucleotides” to several thousand monomeric units. Whenever a polynucleotide or oligonucleotide is represented by a sequence of letters (upper or lower case), such as "ATGCCTG,” it will be understood that the nucleotides are in 5' ⁇ 3' order from left to right.
• the methods comprise hybridizing at least parts of the first and second probe sets to first and second nucleic acid regions of interest in nucleotide molecules of the genetic sample, respectively.
• the hybridization of the probes to the nucleic acid of interest may be performed simultaneously or after contacting the probes to the genetic sample, ligating, amplifying and/or immobilizing the probes.
• the hybridization of the probes to the nucleic acid of interest may be performed simultaneously or before ligating, amplifying, and/or immobilizing the probes.
• a part or full part of the probe may hybridize to a part or full part of the region of interest in single or double stranded nucleotide molecules, protein, or antibody in a sample.
• the region of interest hybridized to the probe may be from 1 to 50 nucleotides, 50 to 1000 nucleotides, 100 to 500 nucleotides, 5, 10, 50, 100, 200 nucleotides or less, or 2, 5, 10, 50, 100, 200, 500, 1000 nucleotides or more.
• Probes may be designed or configured to hybridize perfectly with a target region or molecule, or they may be designed such that a single-base mismatch (e.g., at a single nucleotide polymorphism, or SNP site), or a small number of such mismatches, fails to yield a hybrid of probe and target molecule.
• the first and second labeling probes are hybridized to the first and second nucleic acid regions of interest in nucleotide molecules of the genetic sample, respectively; the first and second tagging probes are hybridized to the first and second nucleic acid regions of interest in nucleotide molecules of the genetic sample, respectively; the first labeling probe is hybridized to a region adjacent to where the first tagging probe is hybridized; and the second labeling probe is hybridized to a region adjacent to where the second tagging probe is hybridized.
• High stringency conditions when used in reference to nucleic acid hybridization, comprise conditions equivalent to binding or hybridization at 68° C in a solution consisting of 5+SSPE, 1% SDS, 5 ⁇ Denhardt's reagent and 100 ⁇ g/ml denatured salmon sperm DNA followed by washing in a solution comprising 0.1+SSPE and 0.1% SDS at 68° C when a probe of about 100 to about 1000 nucleotides in length is employed.
• the probe product may be formed only if the probes within a probe set are correctly hybridized. Therefore, the probe products may be formed with high stringency and high accuracy. Again, the probe products may contain sufficient information for identifying the genomic sequence for which the probe product was designed to interrogate. Therefore, generation and direct quantification of a particular probe product (in this case, by molecular counting) may reflect the abundance of a particular genetic sequence in the originating sample.
• the nucleic acid regions of interest, to which the probes are configured to hybridize to are located in different chromosomes.
• the first nucleic acid region of interest is located in chromosome 21, and the second nucleic acid region of interest is not located in chromosome 21 (e.g., located in chromosome 18).
• Primers are usually single-stranded for maximum efficiency in amplification, but may alternatively be double-stranded. If double-stranded, the primer is usually first treated to separate its strands before being used to prepare extension products. This denaturation step is typically influenced by heat, but may alternatively be carried out using alkali, followed by neutralization.
• a "primer" is complementary to a template, and complexes by hydrogen bonding or hybridization with the template to give a primer/template complex for initiation of synthesis by a polymerase, which is extended by the addition of covalently bonded nucleotides linked at its 3' end complementary to the template in the process of DNA synthesis.
• a “primer pair” as used herein refers to a forward primer and a corresponding reverse primer, having nucleic acid sequences suitable for nucleic acid-based amplification of a target nucleic acid.
• Such primer pairs generally include a first primer having a sequence that is the same or similar to that of a first portion of a target nucleic acid, and a second primer having a sequence that is complementary to a second portion of a target nucleic acid to provide for amplification of the target nucleic acid or a fragment thereof.
• Reference to "first” and “second” primers herein is arbitrary, unless specifically indicated otherwise.
• the nucleic acid region of interest in the nucleotide molecule herein may be amplified by the amplification methods described herein.
• the nucleic acids in a sample may or may not be amplified prior to analysis, using a universal amplification method (e.g., whole genome amplification and whole genome PCR).
• the amplification of the nucleic acid region of interest may be performed simultaneously or after contacting the probes to the genetic sample, ligating, amplifying and/or immobilizing the probes.
• the amplification of the ligated probes may be performed simultaneously or before contacting the probes to the genetic sample, ligating the probes, immobilizing the probes, and/or counting the labels.
• the substrate herein may comprise a binding partner that is configured to contact and bind to a part or full tag in the tagging probe described herein and immobilize the tag and thus the tagging probe comprising the tag.
• the tag of the tagging probe may comprise a corresponding binding partner of the binding partner on the substrate as described herein.
• Immobilization may be performed by hybridizing a part or full tagging probe to a part or full binding partner on the substrate.
• the immobilizing step comprises hybridizing at least a part of the tag or tagging nucleotide sequence to a corresponding nucleotide molecule immobilized on the substrate.
• the corresponding nucleotide molecule is a binding partner of the tag or tagging nucleotide sequence that is configured to hybridize partially or fully to the tag or tagging nucleotide sequence.
• the oligonucleotide or polynucleotide binding partners may be single stranded and may be covalently attached to the substrate, for example, by 5'-end or a 3'-end.
• Immobilization may also be performed by the following exemplary binding partners and binding means: Biotin-oligonucleotide complexed with Avidin, Strepatavidin or Neutravidin; SH-oligonucleotide covalently linked via a disulphide bond to a SH-surface; Amine-oligonucleotide covalently linked to an activated carboxylate or an aldehyde group; Phenylboronic acid (PBA)-oligonucleotide complexed with salicylhydroxamic acid (SHA); Acrydite-oligonucleotide reacted with thiol or silane surface or co-polyemerized with acrylamide monomer to form polyacrylamide, or by other methods known in the art.
• PBA Phenylboronic acid
• SHA salicylhydroxamic acid
• Acrydite-oligonucleotide reacted with thiol or silane surface or co-polyemerized with acrylamide monomer
• surface layers may be composed of a polyelectrolyte multilayer (PEM) structure as shown in U.S. Patent Application Publication No. 2002/025529 .
• the immobilization may be performed by well-known procedures, for example, comprising contacting the probes with the support having binding partners attached for a certain period of time, and after the probes are depleted for the extension, the support with the immobilized extension products is optionally rinsed using a suitable liquid.
• immobilizing probe products onto a substrate may allow for rigorous washing for removing components from the biological sample and the assay, thus reducing background noise and improving accuracy.
• Solid support “support,” “substrate,” and “solid phase support” are used interchangeably and refer to a material or group of materials having a rigid or semi-rigid surface or surfaces.
• at least one surface of the substrate will be substantially flat or have wells, raised regions, pins, etched trenches, or the like.
• the substrate may comprise at least one planar solid phase support (e.g., a glass microscope slide).
• the substrate(s) will take the form of beads, resins, gels, microspheres, or other geometric configurations.
• the substrate according to some embodiments of the present disclosure excludes beads, resins, gels, and/or microspheres.
• the binding partners, the tags, the affinity tags, labels, the probes (e.g., tagging probes and labeling probes), and/or the probe sets described herein may be immobilized on a substrate (1) as an array (2).
• the array has multiple members (3-10) that may or may not have an overlap (6) between the members.
• Each member may have at least an area with no overlap with another member (3-5 and 7-10).
• each member may have different shapes (e.g., circular spots (3-8), triangles (9), and squares (10)) and dimensions.
• a member of an array may have an area about from 1 to 10 7 micron 2 , from 100 to 10 7 micron 2 , from 10 3 to 10 8 micron 2 , from 10 4 to 10 7 micron 2 ; from 10 5 to 10 7 micron 2 ; about 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 or more micron 2 ; and/or about 0.001, 0.01, 0.1, 1, 10, 100, 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 or less micron 2 .
• An image of an exemplary member (8) according to some embodiments of the present invention is shown as item 12.
• two or more members comprising the binding partners, the tags, the affinity tags, labels, the probes may have the same shape and dimension.
• the members of an array comprising the binding partners, tags, affinity tags, labels, tagging probes and/or probe sets configured or used to detect the same genetic variation or a control according to the methods described herein may have the same shapes and dimensions.
• each and every member of the arrays on the substrate may have the same shapes and dimensions.
• the members of an array comprising the binding partners, tags, affinity tags, labels, probes and/or probe sets configured or used to detect different genetic variations and/or controls according to the methods described herein may have the same shapes and dimensions.
• each member of the array may comprise different binding partners, the tags, the affinity tags, labels, the probes, and/or the probe sets.
• two members of the array may be separated by (i) a distance, in which there may be no or only very few binding partners, the tags, the affinity tags, labels, the probes (e.g., tagging probes and labeling probes), and/or the probe sets immobilized, and/or (ii) any separator distinguishing one member from the other (e.g., heightened substrate, any material preventing binding of the binding partners, the tags, the affinity tags, the probes (e.g., tagging probes), and/or the probe sets to the substrate, and any non-probe material between the members).
• the members of the array may be distinguished from each other at least by their locations alone.
• the members of the array may be separated by a distance about from 0 to 10 4 microns, from 0 to 10 3 microns, from 10 2 to 10 4 microns, or from 10 2 to 10 3 microns; about 0, 0.001, 0.1, 1, 2, 3, 4, 5, 10, 50, 100, 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , or 10 8 microns or more; and/or about 0, 0.001, 0.1, 1, 2, 3, 4, 5, 10, 50, 100, 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , or 10 8 microns or less.
• the distance by which two members of the array are separated may be determined by the shortest distance between the edges of the members.
• the distance by which two members, items 3 and 4, of an array (2) are separated is the distance indicated by item n.
• the shortest distance by which the members of the array (2) on a substrate (1) are separated is 0, as the distance by which two members, items 10 and 11, of the array are separated.
• two members of the array may not be separated and may be overlapped (6).
• each member may have at least an area with no overlap with another member (7).
• an array and the members of the array of the binding partners, the tags, the affinity tags, labels, the probes, and/or the probe sets described herein may be located on predetermined locations on the substrate, and the shapes and dimensions of each member of the array and the distance between the members may be predetermined prior to the immobilization.
• the predetermined location herein means a location that is determined or identified prior to the immobilization. For example, the shape and dimension of each member of an array is determined or identified prior to the immobilization.
• the substrate may comprise an array of binding partners, each member of the array comprising the binding patners, such as oligonucleotides or polynucleotides, that are immobilized (e.g., by a chemical bond that would be not broken during the hybridization of probes to the binding partners of the substrate described herein) to a spatially defined region or location; that is, the regions or locations are spatially discrete or separated by a defined region or location on the substrate.
• the substrate may comprise an array, each member of which comprises binding partners binding to a spatially defined region or location.
• Each of the spatially defined locations configured to comprise the binding partners may additionally be "addressable" in that its location and the identity of its immobilized binding partners are known or predetermined, for example, prior to its use, analysis, or attaching to their binding partners in tagging probes and/or probe sets.
• an address of an end-attached part of a probe set is a spatial location, e.g., the planar coordinates of a particular region immobilizing copies of the end-attached part of the probe set.
• end-attached parts of probe sets may be addressed in other ways too, e.g., by color, frequency of micro-transponder, or the like, e.g., Chandler et al, PCT publication WO 97/14028 .
• the labeling probe herein means a probe that comprises or is configured to bind to a label.
• the labeling probe itself may comprise a label or may be modified to comprise or bind to a label.
• the amplified probe herein is defined to be the additional copies of an initial probe produced after amplification of the initial probe as described herein. Accordingly, the amplified probes may have a sequence that is the nucleotide sequences of the initial probes and/or complementary sequence of the nucleotide sequences of the initial probes.
• the amplified probes may contain a sequence that is partial or complete match to the nucleotide sequences of the initial probes.
• Immobilized probe herein is defined to be a probe that is directly or indirectly binding to the substrate by a physical or chemical bond.
• a labeling probe may be immobilized to a substrate indirectly via ligation to a tagging probe immobilized to the substrate described herein.
• a label herein means an organic, naturally occurring, synthetic, artificial, or non-naturally occurring molecule, dye, or moiety having a property or characteristic that is capable of detection and, optionally, of quantitation.
• a label may be directly detectable (e.g., radioisotopes, fluorophores, chemiluminophores, enzymes, colloidal particles, fluorescent substances, Quantum dots or other nanoparticles, nanostructures, metal compounds, organometallic labels, and peptide aptamers); or a label may be indirectly detectable using specific binding partners.
• the fluorescent substances include fluorescent dyes such as fluorescein, phosphor, rhodamine, polymethine dye derivatives, and the like.
• fluorescent dyes such as BODYPY FL (trademark, produced by Molecular Probes, Inc.), FluorePrime (product name, produced by Amersham Pharmacia Biotech, Inc.), Fluoredite (product name, produced by Millipore Corporation), FAM (produced by ABI Inc.), Cy 3 and Cy 5 (produced by Amersham pharmacia), TAMRA (produced by Molecular Probes, Inc.), Pacific Blue, TAMRA, Alexa 488, Alexa 594, Alexa 647, Atto 488, Atto 590, Atto 647N and the like.
• fluorescent dyes such as BODYPY FL (trademark, produced by Molecular Probes, Inc.), FluorePrime (product name, produced by Amersham Pharmacia Biotech, Inc.), Fluoredite (product name, produced by Millipore Corporation), FAM (produced by ABI Inc.), Cy 3 and Cy 5 (produced by Amersham pharmacia), TAMRA (produced by Molecular Probes, Inc.), Pacific Blue, TAMRA, Alexa 488
• the immobilized labels are optically resolvable.
• optically resolvable label or “optically individually resolvable label” herein means a group of labels that may be distinguished from each other by their photonic emission, or other optical properties, for example, after immobilization as described herein. Sometimes, even though the labels may have the same optical and/or spectral emission properties, the immobilized labels may be distinguished from each other spatially. Sometimes, the labels of the same type, which is defined to be labels having the same optical properties, are immobilized on the substrate, for example as a member of an array described herein, at a density and/or spacing such that the individual probe products are resolvable as shown in item 12 of Figure 1 .
• the immobilized labels of the same type are separated by a distance about from 1 to 1000 nm, from 5 to 100 nm, or from 10 to 100 nm; about 100, 150, 200, 250, 300, 350, or 400 nm or more; and/or about 50, 100, 150, 200, 250, 300, 350, or 400 nm or less in all dimensions.
• the density of the probe products and their labels on the substrates may be up to many millions (and up to one billion or more) probe products to be counted per substrate. The ability to count large numbers of probe products containing the labels allows for accurate quantification of nucleic acid sequences.
• the tag or affinity tag examples include a binding partner described herein, unique DNA sequences allowing for sequence-specific capture including natural genomic and/or artificial non-genomic sequence, biotin-streptavidin, His-tags, FLAG octapeptide, click chemistry (e.g., pairs of functional groups that rapidly and selectively react with each other under mild, aqueous conditions), and antibodies (e.g., azide-cycline).
• the immobilizing step comprises hybridizing at least a part of the tag, affinity tag, or tagging nucleotide sequence to a corresponding nucleotide molecule immobilized on the substrate.
• the tag or affinity tag is configured to bind to entities including, but not limited to a bead, a magnetic bead, a microscope slide, a coverslip, a microarray or a molecule.
• the immobilizing step is performed by immobilizing the tags to the predetermined location of the substrate.
• the numbers of different labels immobilized on the substrate and thus the numbers of different immobilized probe products comprising the labels are counted.
• the probe products from each genetic locus are grouped together, and the labels in the immobilized probe products are counted.
• multiple sequences within a genomic locus may be interrogated via the creation of multiple probe product types. For this example, different probe products for the same genomic locus may be combined (possibly via immobilization to a common location of a substrate, e.g., as a member of an array described herein), and the labels in these probe products may be directly counted.
• Different probe products for the same genomic locus may be also separated (possibly via immobilization to different locations of a substrate, e.g., as different members of an array described herein), and the labels in these probe products may be directly counted.
• the substrate may have one or more specific affinity tag in each location on a substrate, e.g., as a member of an array on the substrate.
• probe products for a single genomic locus this may be one probe product type, or may be a set of more than one probe product for a particular genomic locus
• a substrate e.g., as the same member of an array described herein
• the probe products from the first genomic locus will be distinguishable from the probe products from the second genomic locus, based on the presence of different labels used in generating the probe products.
• a set of probe products corresponding to chromosome 21 would be generated, for example with a red fluorophore label, and counted.
• a second set of probe products would also be generated from a reference, or control locus, for example chromosome 18, and counted. This second set of probe products may be generated, for example, with a green fluorophore label.
• these probe products may be prepared such that they are grouped together by locus (in this case chromosome 21 or chromosome 18) and counted separately on a substrate. That is, the probe products corresponding to chromosome 21 may be isolated and counted separately, and the probe products corresponding to chromosome 18 may be isolated and counted separately.
• These probe products may be also prepared in such a way that they are grouped together in the same location of a substrate (e.g., as the same member of an array described herein). In this case, on the same region of a substrate, the probe products bearing a red fluorophore will correspond to chromosome 21, and the probe products with a green fluorophore will correspond to chromosome 18.
• chromosome 21 probe products since all of these probe products are individually resolvable and may therefore be counted very accurately, an increased frequency of chromosome 21 probe products relative to chromosome 18 probe products (even as small as 0.01, 0.1, one or more percent or less) will signify the presence of trisomy 21 in a fetus.
• the probe products for chromosome 18 may serve as a control.
• the counting step comprises reading the substrate in first and second imaging channels that correspond to the first and second labels, respectively, and producing one or more images of the substrate, wherein the first and second labeling probes are resolvable in the one or more images.
• the counting step comprises spatial filtering for image segmentation.
• the counting step comprises watershedding analysis, or a hybrid method for image segmentation.
• the methods may also look at the frequency of different alleles at the same genetic locus (e.g., two alleles of a given single nucleotide polymorphisms).
• the accuracy of these methods may detect very small changes in frequency (e.g., as low as about 10, 5, 4, 3, 2, 1, 0.5, 0.1 or 0.01 % or less).
• a blood sample will contain a very dilute genetic signature from the donated organ. This signature may be the presence of an allele that is not in the recipient of the donated organ's genome.
• the methods may detect very small deviations in allele frequency (e.g., as low as about 10, 5, 4, 3, 2, 1, 0.5, 0.1 or 0.01 % or less) and may identify the presence of donor DNA in a host sample (e.g., blood sample).
• a host sample e.g., blood sample
• An unhealthy transplanted organ may result in elevated levels of donor DNA in the host blood - a rise of only a few percent (e.g., as low as about 10, 5, 4, 3, 2, 1, 0.5, 0.1 or 0.01 % or less).
• the methods may be sensitive enough to identify changes in allele frequency with the necessary sensitivity, and therefore may accurately determine the presence and changing amounts of donor DNA in host blood.
• the methods comprise comparing the first and second numbers to determine the genetic variation in the genetic sample.
• the comparing step comprises obtaining an estimate of a relative number of the nucleotide molecules having the first and second nucleic acid regions of interest.
• the methods of the present disclosure may comprise labeling the first and second labeling probes with the first and second labels, respectively, prior to the contacting step (e.g., during manufacturing the probes). Labeling the probe may be performed simultaneously or after contacting the probes to the genetic sample, hybridizing, ligating, amplifying and/or immobilizing the probes. Moreover, labeling the probe may be performed simultaneously or before contacting the probes to the genetic sample, hybridizing, ligating, amplifying, and/or immobilizing the probes. Labeling a probe may comprise adding, immobilizing, or binding a label to the probe by a physical or chemical bond. Labels may be placed anywhere within the sequence of a probe, including at the 5' or 3'-end.
• the probe sets herein may be designed to have tags according to the predetermined locations to which the tags are to be immobilized.
• the tags in all probe sets configured to detect a genetic variation are the same and are configured to be immobilized to same locations on the substrate directly or indirectly.
• the first and second tags are the same, and each of the rest of the tags is different from the first or second tag.
• each of a group of members of the array of multiple predetermined locations on a substrate may have a unique tag to be immobilized.
• the probe sets are amplified, and labeled probe sets are produced during the process of amplification.
• Each of the labeling probes may comprise a forward or reverse priming sequence
• each of the tagging probes may comprise a corresponding reverse or forward priming sequence and a tagging nucleotide sequence as a tag.
• the forward and reverse priming sequences are the sequences that are configured to hybridize to the corresponding forward and reverse primers, respectively.
• the amplified tagging nucleotide sequences of the tagging probes are immobilized to a pre-determined location on a substrate, wherein the amplified tagging nucleotide sequences of the first and second tagging probes are the first and second tags.
• the first and second tags are the same and/or are configured to bind to the same location on the substrate.
• the first and second tags are different and/or are configured to bind to different locations on the substrate.
• the method comprises counting numbers of the labels in the amplified probes and/or probe sets immobilized on the substrate. For example, the first number is the number of the first label in the amplified first probe set immobilized to the substrate, and the second number is the number of the second label in the amplified second probe set immobilized to the substrate.
• the probe sets according to some embodiments may be amplified, and labeled probe sets may be produced using labeled reverse primers without using a forward primer.
• each of the labeling probes may comprise a reverse priming sequence
• each of the tagging probes may comprise a tagging nucleotide sequence as a tag.
• the amplifying step may comprise amplifying (i) the ligated first labeling and tagging probes with a first reverse primer hybridizing to a first reverse priming sequence of the first labeling probe, wherein the first reverse primer comprises the first label, and (ii) the ligated second labeling and tagging probes with a second reverse primer hybridizing to a second reverse priming sequence of the second labeling probe, wherein the second reverse primer comprises the second label.
• the amplified tagging nucleotide sequences of the tagging probes are immobilized to a pre-determined location on a substrate, wherein the amplified tagging nucleotide sequences of the first and second tagging probes are the first and second tags.
• the first number is the number of the first label in the amplified first probe set immobilized to the substrate
• the second number is the number of the second label in the amplified second probe set immobilized to the substrate.
• the ligated probe sets may be produced using a ligase chain reaction.
• the method described herein comprises contacting third and fourth probe sets to the genetic sample, wherein the third probe set comprises a third labeling probe and a third tagging probe, and the fourth probe set comprises a fourth labeling probe and a fourth tagging probe.
• the method may further comprise hybridizing the first and second probe sets to first and second sense nucleic acid strands of interest in single stranded nucleotide molecules from the double stranded nucleotide molecules of the genetic sample, respectively; and hybridizing the third and fourth probe sets to anti-sense nucleic acid strands of the first and second sense nucleic acid strands of interest, respectively.
• the ligase chain reaction may comprise hybridizing non-ligated first, second, third and fourth probe sets to the ligated third, fourth, first, and second probe sets, respectively, and ligating at least (i) the first labeling probe and the first tagging probe, (ii) the second labeling probe and the second tagging probe, (iii) the third labeling probe and the third tagging probe, and (iv) the fourth labeling probe and the fourth tagging probe of the non-ligated probe sets.
• the method may further comprise immobilizing the tagging probes to the pre-determined location on a substrate, wherein the first, second, third and fourth labeling probes ligated to the immobilized first, second, third and fourth tagging probes, respectively, comprise first, second, third and fourth labels, respectively; the immobilized labels are optically resolvable; the immobilized first, second, third and fourth tagging probes comprise first, second, third and fourth tags, respectively, and the immobilizing step is performed by immobilizing the tags to the predetermined location.
• the method may further comprise counting (i) the first sum of the first and third labels immobilized to the substrate, and (ii) the second sum of the second and fourth labels immobilized to the substrate, and comparing the first and second sums to determine the genetic variation in the genetic sample.
• the method further comprises labeling the first, second, third and fourth labeling probes with the first, second, third and fourth labels, respectively, prior to the contacting step.
• the first and third labels are the same, and the second and fourth labels are the same.
• the method may further comprise hybridizing the first and second probe sets to first and second sense nucleic acid strands of interest, respectively, in single stranded nucleotide molecules from double stranded nucleotide molecules of the genetic sample; and hybridizing at least parts of the third and fourth probe sets to anti-sense nucleic acid strands of the first and second sense nucleic acid strands of interest, respectively; producing ligated first, second, third, and fourth probe sets by ligating (i) the first labeling probe and the first tagging probe, (ii) the second labeling probe and the second tagging probe, (iii) the third labeling probe and the third tagging probe, and (iv) the fourth labeling probe and the fourth tagging probe.
• the method may further comprise performing a ligase chain reaction.
• the ligase chain reaction comprises hybridizing at least parts of the non-ligated first, second, third and fourth probe sets to the ligated third, fourth, first, and second probe sets, respectively, and ligating (i) the first labeling probe and the first tagging probe, (ii) the second labeling probe and the second tagging probe, (iii) the third labeling probe and the third tagging probe, and (iv) the fourth labeling probe and the fourth tagging probe of the non-ligated probe set.
• the ligated first and second labeling probes are at the 3'-end of the first and second ligated probe set and comprise first and second reverse priming sequences hybridizing to the first and second reverse primers, respectively.
• the first and second reverse primers comprise the first and second labels.
• the ligated first and second tagging probes are at the 5'-end of the first and second ligated probe set.
• the ligated first and second tagging probes are at the 5'-end of the first and second ligated probe set and comprise first and second corresponding forward priming sequences hybridizing to the first and second forward primers, respectively.
• the method herein comprises digesting double stranded molecules in the sample to produce single stranded molecules.
• the amplifying step comprises contacting an exonuclease to the amplified probe and/or probe set, and digesting the amplified probe and/or probe set from the 5'-end of one strand of the double stranded amplified probe and/or probe set.
• the amplifying step comprises contacting an exonuclease to the amplified probe in a probe set, and digesting the amplified probe set from the 5'-end of one strand of the double stranded amplified probe set.
• the one strand of the amplified probe and probe set contacting the exonuclease does not have any label at the 5'-end.
• the contacting of the exonuclease to the unlabeled double stranded probes may digest the unlabeled strand from the 5'-end producing single stranded probes.
• the 5'-end of the amplified probe set comprising the label at the 5'-end may be protected from exonuclease digestion.
• the method may detect from 1 to 100, from 1 to 50, from 2 to 40, or from 5 to 10 genetic variations; 2, 3, 4, 5, 6, 7, 8, 9, 10 or more genetic variations; and 100, 50, 30, 20, 10 or less genetic variations.
• the method described herein may detect x number of genetic variations using at least (x+1) number of different probe sets. In these embodiments, a number of labels from one type of probe sets may be compared with one or more numbers of labels from the rest of the different types of probe sets.
• the method described herein may detect genetic variation in a continuous manner across the entire genome at various resolutions, for example, at 300,000 base resolution such that 100 distributed variations across all chromosomes are separately interrogated and quantified. In additional embodiments, the base resolution is in the range of one or ten to 100 thousand nucleotides up to one million, ten million, or 100 million or more nucleotides.
• the method according to some embodiments may detect at least two genetic variations.
• the method described herein may further comprise contacting a fifth probe set to the genetic sample, wherein the fifth probe set comprises a fifth labeling probe and a fifth tagging probe.
• the method may further comprise hybridizing at least a part of the fifth probe set to the third nucleic acid region of interest in nucleotide molecules of the genetic sample, wherein the third nucleic acid region of interest is different from the first and second nucleic acid regions of interest.
• the method may further comprise ligating the fifth probe set at least by ligating the fifth labeling probe and the fifth tagging probe.
• the method may further comprise amplifying the ligated probe sets.
• the method may further comprise immobilizing each of the tagging probe to a pre-determined location on a substrate, wherein the fifth labeling probe and/or the amplified labeling probe thereof ligated to the immobilized tagging probe comprise a fifth label, the fifth label is different from the first and second labels, the immobilized labels are optically resolvable, the immobilized fifth tagging probe and/or the amplified tagging probe thereof comprise a fifth tag, and the immobilizing step is performed by immobilizing the tags to the predetermined location.
• the method may comprise counting a third number of the fifth label immobilized to the substrate, and comparing the third number to the first and/or second number(s) to determine the second genetic variation in the genetic sample.
• the subject may be a pregnant subject
• the first genetic variation is trisomy 21 in the fetus of the pregnant subject
• the second genetic variation is selected from the group consisting of trisomy 13, trisomy 18, aneuploidy of X, and aneuploidy of Y in the fetus of the pregnant subject.
• the method according to some embodiments may detect at least three genetic variations.
• the method described herein further comprises contacting a sixth probe set to the genetic sample, wherein the sixth probe set comprises a sixth labeling probe and a sixth tagging probe.
• the method may further comprise hybridizing at least a part of the sixth probe set to the fourth nucleic acid region of interest in nucleotide molecules of the genetic sample, wherein the fourth nucleic acid region of interest is different from the first, second, and third nucleic acid regions of interest.
• the method may further comprise ligating the sixth probe set at least by ligating the sixth labeling probe and the sixth tagging probe.
• the method may further comprise amplifying the ligated probe sets.
• the method may further comprise immobilizing each of the tagging probes to a pre-determined location on a substrate, wherein the sixth labeling probe and/or the amplified labeling probe thereof ligated to the immobilized tagging probe comprise a sixth label, the sixth label is different from the first and second labels, the immobilized labels are optically resolvable, the immobilized sixth tagging probe and/or the amplified tagging probe thereof comprise a sixth tag, and the immobilizing step is performed by immobilizing the tags to the predetermined location.
• the method may further comprise counting a fourth number of the sixth label immobilized to the substrate, and comparing the fourth number to the first, second and/or third number to determine the third genetic variation in the genetic sample.
• the method may according to some embodiments detect at least four genetic variations.
• the method described herein further comprises contacting a seventh probe set to the genetic sample, wherein the seventh probe set comprises a seventh labeling probe and a seventh tagging probe.
• the method may further comprise hybridizing at least a part of the seventh probe set to the fifth nucleic acid region of interest in nucleotide molecules of the genetic sample, wherein the fifth nucleic acid region of interest is different from the first, second, third and fourth nucleic acid regions of interest.
• the method may further comprise ligating the seventh probe set at least by ligating the seventh labeling probe and the seventh tagging probe.
• the method may further comprise optionally amplifying the ligated probe sets.
• the method according to some embodiments may detect at least five genetic variations.
• the method described herein further comprises contacting an eighth probe set to the genetic sample, wherein the eighth probe set comprises an eighth labeling probe and an eighth tagging probe.
• the method may further comprise hybridizing at least a part of the eighth probe set to the sixth nucleic acid region of interest in nucleotide molecules of the genetic sample, wherein the sixth nucleic acid region of interest is different from the first, second, third, fourth, and fifth nucleic acid regions of interest.
• the method may further comprise ligating the eighth probe set at least by ligating the eighth labeling probe and the eighth tagging probe.
• the method may further comprise amplifying the ligated probe sets.
• the subject is a pregnant subject
• the first, second, third, fourth, and fifth genetic variations are trisomy 13, trisomy 18, trisomy 21, aneuploidy X, and aneuploidy Y in the fetus of the pregnant subject.
• the subject is a pregnant subject
• the genetic variation is trisomy 21 in the fetus of the pregnant subject
• the first nucleic acid region of interest is located in chromosome 21
• the second nucleic acid region of interest is not located in the chromosome 21.
• the subject is a pregnant subject
• the genetic variation is trisomy 21 in the fetus of the pregnant subject
• the first nucleic acid region of interest is located in chromosome 21
• the second nucleic acid region of interest is located in chromosome 18.
• the probe set herein may comprise two, three, four, five or more labeling probes, and/or two, three, four, five or more labels.
• the method described herein may further comprise the first and second probe sets further comprise third and fourth labeling probes, respectively; the immobilized first probe set and/or amplified first probe set further comprise a ninth label in the third labeling probe and/or amplified product thereof; and the immobilized second probe set and/or amplified second probe set further comprise a tenth label in the fourth labeling probe and/or amplified product thereof.
• this method may be used to confirm the number counted for the first and second labels.
• this method may be used to improve the accuracy of detection labels immobilized to each of the nucleic acid regions of interest. For example, using multiple labels would be brighter than using one label, and therefore multiple labels may be more easily detected than one label.
• the immobilized first probe set and/or amplified first probe set further comprise an eleventh label in the labeling probe
• the immobilized second probe set and/or amplified second probe set further comprises a twelfth label that is different from the eleventh label in the labeling probe.
• the counting step further comprises counting numbers of the eleventh and twelfth labels immobilized on the substrate.
• the method described herein may be performed with a control sample.
• the method may further comprise repeating the steps with a control sample different from the genetic sample from the subject.
• the method may further comprise counting control numbers of the labels immobilized to the substrate, and comparing the control numbers to the first, second, third, fourth, fifth and/or sixth number to confirm the genetic variation in the genetic sample.
• the subject is a pregnant subject
• the genetic variation is a genetic variation in the fetus of the pregnant subject.
• the method may use a Single Nucleotide Polymorphism (SNP) site to determine whether the proportion (e.g., concentration, and number percentage based on the number of nucleotide molecules in the sample) of fetal material (e.g., the fetal fraction) is sufficient so that the genetic variation of the fetus may be detected from a sample from the pregnant subject with a reasonable statistical significance.
• SNP Single Nucleotide Polymorphism
• the method may further comprise contacting maternal and paternal probe sets to the genetic sample, wherein the maternal probe set comprises a maternal labeling probe and a maternal tagging probe, and the paternal probe set comprises a paternal labeling probe and a paternal tagging probe.
• the method may further comprise hybridizing at least a part of each of the maternal and paternal probe sets to a nucleic acid region of interest in nucleotide molecules of the genetic sample, the nucleic acid region of interest comprising a predetermined SNP site, wherein the at least a part of the maternal probe set hybridizes to a first allele at the SNP site, the at least a part of the paternal probe set hybridizes to a second allele at the SNP site, and the first and second alleles are different from each other.
• the method may further comprise ligating the maternal and paternal probe sets at least by ligating (i) the maternal labeling and tagging probes, and (ii) the paternal labeling and tagging probes.
• the method may further comprise amplifying the ligated probes.
• the method may further comprise immobilizing the tagging probes to a pre-determined location on a substrate, wherein the maternal and paternal labeling probes and/or the amplified labeling probes thereof ligated to the immobilized tagging probes comprise maternal and paternal labels, respectively; the maternal and paternal labels are different, and the immobilized labels are optically resolvable.
• the method may further comprise counting the numbers of the maternal and paternal labels, and determining whether a proportion of a fetal material in the genetic sample is sufficient to detect the genetic variation in the fetus based on the numbers of the maternal and paternal labels.
• the method may further comprise determining the proportion of the fetal material in the genetic sample.
• the method may further comprise hybridizing at least a part of each of the allele A and allele B probe sets to a nucleic acid region of interest in nucleotide molecules of the genetic sample, the nucleic acid region of interest comprising a predetermined single nucleotide polymorphism (SNP) site for which a maternal allelic profile (i.e., genotype) differs from a fetal allelic profile at the SNP site
• SNP single nucleotide polymorphism
• maternal allelic composition may be AA and fetal allelic composition may be AB, or BB.
• maternal allelic composition may be AB and fetal allelic composition may be AA, or BB.
• the method may further comprise ligating the allele A and allele B probe sets at least by ligating (i) the allele A labeling and tagging probes, and (ii) the allele B labeling and tagging probes.
• the method may further comprise amplifying the ligated probe sets.
• the method may further comprise immobilizing the tagging probes to a pre-determined location on a substrate, wherein the allele A and allele B labeling probes and/or the amplified labeling probes thereof ligated to the immobilized tagging probes comprise allele A and allele B labels, respectively, the allele A and allele B labels are different, and the immobilized labels are optically resolvable.
• the method may further comprise counting the numbers of the allele A and allele B labels, and determining whether a proportion of a fetal material in the genetic sample is sufficient to detect the genetic variation in the fetus based on the numbers of the allele A and allele B labels.
• the method may further comprise determining the proportion of the fetal material in the genetic sample.
• the method may further comprise contacting maternal and paternal probe sets to the genetic sample, wherein the maternal probe set comprises a maternal labeling probe and a maternal tagging probe, and the paternal probe set comprises a paternal labeling probe and a paternal tagging probe.
• the method may further comprise hybridizing at least parts of the maternal and paternal probe sets to maternal and paternal nucleic acid regions of interest in nucleotide molecules of the genetic sample, respectively, wherein the paternal nucleic acid region of interest is located in the Y chromosome, and the maternal nucleic acid region of interest is not located in the Y chromosome.
• the method may further comprise ligating the maternal and paternal probe sets at least by ligating (i) the maternal labeling and tagging probes, and (ii) the paternal labeling and tagging probes.
• the method may further comprise amplifying the ligated probes.
• the method may further comprise nucleic acid region of interest comprising a predetermined single nucleotide polymorphism (SNP) site containing more than one SNP, for example two or three SNPs.
• SNP site may contain SNPs with high linkage disequilibrium such that labeling and tagging probes are configured to take advantage of the improved energetics of multiple SNP matches or mismatches versus only one.
• the method may further comprise immobilizing the tagging probes to a pre-determined location on a substrate, wherein the maternal and paternal labeling probes and/or the amplified labeling probes thereof ligated to the immobilized tagging probes comprise maternal and paternal labels, respectively, the maternal and paternal labels are different, and the immobilized labels are optically resolvable.
• the method may further comprise counting the numbers of the maternal and paternal labels, and determining whether a proportion of a fetal material in the genetic sample is sufficient to detect the genetic variation in the fetus based on the numbers of the maternal and paternal labels.
• the method may further comprise determining the proportion of the fetal material in the genetic sample.
• genetic variations e.g., single base deletion, microsatellite, and small insertions
• SNP site e.g., single base deletion, microsatellite, and small insertions
• the probe set described herein may comprise three or more probes, including at least one probe between the labeling and tagging probes.
• the first and second probe sets further comprises first and second gap probes, respectively; the first gap probe hybridizes to a region between the regions where the first labeling probe and the first tagging probe hybridize; the second gap probe hybridizes to a region between the regions where the second labeling probe and the second tagging probe hybridize.
• the method may further comprise the ligating step comprises ligating at least (i) the first labeling probe, the first tagging probe, and the first gap probe, and (ii) the second labeling probe, the second tagging probe, and the second gap probe.
• the gap probe may comprise a label.
• the first and second labeling probes are hybridized to the first and second nucleic acid regions of interest in nucleotide molecules of the genetic sample, respectively; the first and second tagging probes are hybridized to the first and second nucleic acid regions of interest in nucleotide molecules of the genetic sample, respectively; the first and second gap probes are hybridized to the first and second nucleic acid regions of interest in nucleotide molecules of the genetic sample, respectively.
• nucleotides there are from 0 to 100 nucleotides, 1 to 100 nucleotides, 2 to 50 nucleotides; 3 to 30 nucleotides, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 150, or 200 or more; or 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 25, 35, 45, 55, 110, 160, or 300 or less between the regions where the first labeling probe and tagging probes are hybridized; and there are from 0 to 100 nucleotides, 1 to 100 nucleotides, 2 to 50 nucleotides; 3 to 30 nucleotides, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 150, or 200 nucleotides or more; or 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 25, 35, 45, 55, 110, 160, or 300 nucleotides or less between the regions where the second labeling probe and tagging probes are hybridized.
• the gap probe between a labeling probe and a tagging probe may have a length from 0 to 100 nucleotides, 1 to 100 nucleotides, 2 to 50 nucleotides; 3 to 30 nucleotides, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 150, or 200 or more; or 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 25, 35, 45, 55, 110, 160, or 300 or less.
• the probe set described herein may comprise a spacer ligated and/or conjugated to the labeling probe and the tagging probe.
• the spacer may or may not comprise oligonucleotides.
• the spacer may comprise an isolated, purified, naturally-occurring, or non-naturally occurring material, including oligonucleotide of any length (e.g., 5, 10, 20, 30, 40, 50, 100, or 150 nucleotides or less).
• the probe may be in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification.
• the first labeling and tagging probes are conjugated by a first spacer
• the second labeling and tagging probes are conjugated by a second spacer
• the first and second spacers are not hybridized to the nucleotide molecules of the genetic sample.
• the method further comprises digesting the hybridized genetic sample with an enzyme, and breaking a bond in the first and second spacers after the digestion.
• the method described herein excludes identifying a sequence in the nucleotide molecules of the genetic sample, and/or sequencing of the nucleic acid region(s) of interest and/or the probes.
• the method excluding sequencing of the probes includes excluding sequencing a barcode and/or affinity tag in a tagging probe.
• the immobilized probe sets to detect different genetic variations, nucleotide regions of interest, and/or peptides of interest need not be detected or scanned separately because sequencing is not required in the methods described herein.
• the numbers of different labels immobilized to the substrate were counted simultaneously (e.g., by a single scanning and/or imaging), and thus the numbers of different labels were not separately counted.
• the method described herein excludes bulk array readout or analog quantification.
• the bulk array readout herein means a single measurement that measures the cumulative, combined signal from multiple labels of a single type, optionally combined with a second measurement of the cumulative, combined signal from numerous labels of a second type, without resolving a signal from each label. A result is drawn from the combination of the one or more such measurements in which the individual labels are not resolved.
• the method described herein may include a single measurement that measures the same labels, different labels of the same type, and/or labels of the same type in which the individual labels are resolved.
• the method described herein may exclude analog quantification and may employ digital quantification, in which only the number of labels is determined (ascertained through measurements of individual label intensity and shape), and not the cumulative or combined optical intensity of the labels.
• intensity and/or single-to-noise is used as a method of identifying single labels.
• dye molecules or other optical labels are in close proximity, they are often impossible to discriminate with fluorescence-based imaging due to the intrinsic limit of the diffraction of light. That is, two labels that are close together will be indistinguishable with no visible gap between them.
• One exemplary method for determining the number of labels at a given location is to examine the relative signal and/or signal-to-noise compared to locations known to have a single fluor. Two or more labels will usually emit a brighter signal (and one that can more clearly be differentiated from the background) than will a single fluor.
• Figure 2 shows the normalized histogram of signal intensity measured from both single label samples and multi-label antibodies (both Alexa 546; verified through bleach profiles). The two populations were clearly separable, and multiple labels may be clearly distinguished from single labels.
• blinking behavior may be used as a method of identifying single labels.
• Many dye molecules are known to temporarily go into a dark state (e.g., Burnette et al., Proc. Natl. Acad. Sci. USA (2011) 108: 21081-21086 ). This produces a blinking effect, where a label will go through one or more steps of bright-dark-bright. The length and number of these dark periods may vary.
• the current invention uses this blinking behavior to discriminate one label from two or more labels that may appear similar in diffraction limited imaging. If there are multiple labels present, it is unlikely the signal will completely disappear during the blinking. More likely is that the intensity will fall as one of the labels goes dark, but the others do not.
• the probability of all the labels blinking simultaneously may be calculated based on the specific blinking characteristics of a dye.
• the counting step of the method described herein may further comprise calibrating and/or confirming the counted numbers by (i) repeating some or all the steps of the methods (e.g., steps including the contacting, binding, hybridizing, ligating, amplifying, and/or immobilizing) described herein with a different probe set(s) configured to bind and/or hybridize to the same nucleotide and/or peptide region(s) of interest or a different region(s) in the same chromosome of interest, and (ii) averaging the counted numbers of labels in the probe sets bound and/or hybridized to the same a nucleotide and/or peptide region of interest or to the same chromosome of interest.
• steps of the methods e.g., steps including the contacting, binding, hybridizing, ligating, amplifying, and/or immobilizing
• a different probe set(s) configured to bind and/or hybridize to the same nucleotide and/or peptide
• the averaging step may be performed before the comparing step so that the averaged counted numbers of labels in a group of different probe sets that bind and/or hybridize to the same nucleotide and/or peptide region of interest are compared, instead of the counted numbers of the labels in the individual probe sets.
• the method described herein may further comprise calibrating and/or confirming the detection of the genetic variation by (i) repeating some or all the steps of the methods (e.g., steps including the contacting, binding, hybridizing, ligating, amplifying, immobilizing, and/or counting) described herein with different probe sets configured to bind and/or hybridize to control regions that does not have any known genetic variation, and (ii) averaging the counted numbers of labels in the probe sets bound and/or hybridized to the control regions.
• steps of the methods e.g., steps including the contacting, binding, hybridizing, ligating, amplifying, immobilizing, and/or counting
• probe products may be labeled with more than one type of fluorophore such that the spectral signature is more complex.
• probe products may always carry a universal fluor, e.g., Alexa647, and a locus-specific fluorophore, e.g., Alexa 555 for locus 1 and Alexa 594 for locus 2. Since contaminants will rarely yield the signature of two fluors, this may further increase the confidence of contamination rejection. Implementation would involve imaging in three or more channels in this example such that the presence or absence of each fluor may be ascertained, by the aforementioned goodness-of-fit method comparing test to reference, yielding calls of locus 1, locus 2 or not a locus product.
• spectral modifiers may also be used to increase spectral information and uniqueness, including FRET pairs that shift the color when in close proximity or other moieties.
• a method may be used to detect a genetic variation in peptide or proteins.
• the methods may comprise contacting first and second probe sets to the genetic sample, wherein the first probe set comprises a first labeling probe and a first tagging probe, and the second probe set comprises a second labeling probe and a second tagging probe.
• the methods may further comprise binding the probe sets to peptide regions of interest by a physical or chemical bond, in place of the hybridizing step described herein in the case of detecting the genetic variation in nucleic acid molecules.
• the methods may further comprise binding at least parts of the first and second probe sets to first and second peptide regions of interest in a peptide of protein of the genetic sample, respectively.
• the binding may be performed by having a binder in at least one probe in the probe set that specifically binds to the peptide region of interest.
• the test system includes a series of modules, some of which are optional or may be repeated depending on the results of earlier modules.
• the test may comprise: (1) receiving a requisition, e.g., from an ordering clinician or physician, (2) receiving a patient sample, (3) performing an assay including quality controls on that sample resulting in a assay-product on an appropriate imaging substrate (e.g., contacting, binding, and/or hybridizing probes to a sample, ligating the probes, optionally amplifying the ligated probes, and immobilizing the probes to a substrate as described herein), (4) imaging the substrate in one or more spectral channels, (5) analyzing image data, (6) performing statistical calculations (e.g., comparing the first and second numbers to determine the genetic variation in the genetic sample), (7) creating and approving the clinical report, and (8) returning the report to the ordering clinician or physician.
• an imaging substrate e.g., contacting, binding, and/or hybridizing probes to a sample, ligating the probes
• tags, affinity tags, and/or tagging probes in the probe products, ligated probe set, or labeled molecule to be immobilized to the substrate may be uniquely designed for every assay and every assay product, all of the parallel assay products may be localized, imaged and quantitated at different physical locations on the imaging substrate.
• the same assay or method (or some of their steps) using the same probes and/or detecting the same genetic variation or control may be performed on multiple samples simultaneously either in the same or different modules (e.g., testing tube) described herein.
• Assays and methods (or some of their steps) using different probes and/or detecting different genetic variations or controls may be performed on single or multiple sample(s) simultaneously either in the same or different modules (e.g., testing tube).
• testing of samples that fail to confirm at least the minimum input fetal fraction may be terminated before additional imaging and analysis takes place. Conversely, if the fetal fraction is above the minimum threshold, further imaging (step 4 of the test) of the genomic targets (e.g., chromosome 21, 18 or 13) may proceed followed by additional analysis (step 5 of the test). Other criteria may also be used and tested.
• the genomic targets e.g., chromosome 21, 18 or 13
• a single or multiple locations on the substrate used to interrogate SNPs may be imaged and analyzed (e.g., in groups of one, two, three, four, five, ten, twenty, fifty or less and/or one, two, three, four, five, ten, twenty, fifty or more) until an informative SNP is detected.
• imaged and analyzed e.g., in groups of one, two, three, four, five, ten, twenty, fifty or less and/or one, two, three, four, five, ten, twenty, fifty or more.
• Determining the fetal fraction of a sample may aide other aspects of the system beyond terminating tests for which the portion of fetal fraction in a sample is inadequate. For example, if the fetal fraction is high (e.g., 20%) then for a given statistical power, the number of counts required per genetic target (e.g., chr21) will be lower; if the fetal fraction is low (e.g., 1%) then for the same statistical power, a very high number of counts is required per genomic target to reach the same statistical significance.
• the fetal fraction is high (e.g. 20%) then for a given statistical power, the number of counts required per genetic target (e.g., chr21) will be lower; if the fetal fraction is low (e.g., 1%) then for the same statistical power, a very high number of counts is required per genomic target to reach the same statistical significance.
• Steps (4) and (5) of the test above may be repeated further for quality control purposes, including assessment of background levels of fluors on the imaging substrate, contaminating moieties, positive controls, or other causes of copy number variation beyond the immediate test (e.g., cancer in the mother or fetus, fetal chimeraism, twinning).
• image analysis may be real-time, and does not require completion of the entire imaging run before generating results (unlike DNA sequencing methods), intermediate results may dictate next steps from a decision tree, and tailor the test for ideal performance on an individual sample.
• Quality control may also encompass verification that the sample is of acceptable quality and present, the imaging substrate is properly configured, that the assay product is present and/or at the correct concentration or density, that there is acceptable levels of contamination, that the imaging instrument is functional and that analysis is yielding proper results, all feeding in to a final test report for review by the clinical team.
• the test above comprises one or more of the following steps: (1) receiving a requisition (from, for example, an ordering clinician or physician), (2) receiving a patient sample, (3) performing an assay (including a allele-specific portion, genomic target portion and quality controls) on that sample resulting in a assay-product-containing imaging substrate, (4-1) imaging the allele-specific region of the substrate in one or more spectral channels, (5-1) analyzing allele-specific image data to compute the fetal fraction, (pending sufficient fetal fraction) (4-2) imaging the genomic target region of the substrate in one or more spectral channels, (5-2) analyzing genomic target region image data to compute the copy number state of the genomic targets, (4-3) imaging the quality control region of the substrate in one or more spectral channels, (5-3) analyzing quality control image data to compute validate and verify the test, (6) performing statistical calculations, (7) creating and approving the clinical report, and (8) sending the report back to the ordering clinician or physician.
• an assay including a allele-specific portion, genomic target portion
• Figure 21 is an implementation of an assay for quantifying genomic copy number at two genomic loci.
• 105 and 106 are target molecules.
• 105 contains sequence corresponding to the first genomic locus "Locus 1" interrogated for copy number (example, chromosome 21)
• 106 contains sequence corresponding the second genomic locus "Locus 2" interrogated for copy number (example, chromosome 18).
• Figure 21 contains an example of one probe set per genomic locus, but multiple probe sets can be designed to interrogate multiple regions within a genomic locus. For example, more than 10, or more than 100, or more than 500 probe sets may be designed that correspond to chromosome 21.
• Figure 21 illustrates only a single probe set for each genomic locus, but importantly this method allows for multiple probe sets for each genomic locus.
• Figure 21 also illustrates a single hybridization event between a target molecule and a probe set.
• target molecules there will be multiple target molecules present in an assay sample. Many target molecules will contain the necessary sequences for hybridization to a probe set, and formation of a probe product. Different target molecules may hybridize to probe sets, as certain target molecules will bear genetic polymorphisms.
• target molecules that arise from genomic DNA may have a random assortment of molecule sizes, as well various beginning and ending sequences. In essence, there are multiple target molecules that may hybridize to a given probe set. In a single assay, multiple copies of a given probe set are added. Therefore, in a single assay up to thousands, or hundreds of thousands, or millions of specific probe products may be formed.
• probe sets may be designed that target "Locus 1,” containing unique probe sequences but the same label type "A.”
• many probe sets may be designed that target "Locus 2,” containing unique probe sequences but the same label type "B.”
• the affinity tags for the many probe sets for Locus 1 may be identical or unique
• the affinity tags for the many probe sets for Locus 2 may be identical or unique.
• One or more probe sets are added to target molecules in a single vessel and exposed to sequence-specific hybridization conditions.
• the three probes (e.g., 101, 102, 103) are hybridized (or attached via a similar probe-target interaction) to the target molecule (105) such there are no gaps in between the probes on the target molecule. That is, the probes from the probe set are adjacent to one another and ligation competent.
• Figure 23 depicts a modification of the general procedure described in Figure 21 .
• Figure 23 depicts two probe sets, one probe set for Locus 1 and one probe set for Locus 2, although as aforementioned, multiple probes sets may be designed for each genomic locus.
• 307 and 314 are target molecules corresponding to Locus 1 and Locus 2, respectively.
• a first probe set contains member probes 302, 303, 305.
• 302 contains a label (301) of type "A.”
• 305 contains an affinity tag (306) which may be used for isolation and identification of the probe product.
• a second probe set with member probes 309, 310, 312 carries respective features as in the first probe set.
• the purpose of this assay type is to be able to accurately quantify the frequency of Allele 1 and Allele 2 in a sample.
• the probe 804 contains one or more labels (803) of type "C.” Therefore, probe products will contain a combination of labels. For Allele 1, probe products will contain labels of type "A” and type "C,” whereas probe products from Allele 2 will contain labels of type "B” and type "C.”
• a first probe set contains member probes 902, 905.
• 902 contains a label (901) of type "A.”
• Item 905 contains an affinity tag (906) which may be used for isolation and identification of the probe product.
• a second probe set with member probes 909, 905 carries respective features as in the first probe set. 905 is identical for both probe sets. However, 909 contains a label (908) of type "B,” distinguishable from type "A.”
• 902 and 909 contain sequences that are nearly identical, and differ by only one nucleotide in the sequence. Therefore, hybridization sequences of these two probes contain complementary regions for Allele 1 (902), and Allele 2 (909).
• each hybridization domain on 902 and 909, as well as experimental hybridization conditions are designed such that probe 902 will only hybridize to Allele 1 and probe 909 will only hybridize to Allele 2.
• the purpose of this assay type is to be able to accurately quantify the frequency of Allele 1 and Allele 2 in a sample.
• Probes 902 and 905 hybridize to sequences corresponding to Allele 1, such that there is a "gap" on the target molecule consisting of one or more nucleotides between hybridized probes 902 and 905.
• a DNA polymerase or other enzyme may be used to synthesize a new polynucleotide species (904) that covalently joins 902 and 905. That is, the probe product formed in this example is a single contiguous nucleic acid molecule with a sequence corresponding to Allele 1, and bearing the labels and/or affinity tags above. Additionally, 904 may contain one or more labels of type "C," possibly as a result of incorporation of a nucleotide bearing a label of type "C.” This example also conveys to the probe product formed for Allele 2, containing probes 909 and 905.
• 1006 and 1007 are target molecules corresponding to Allele 1 and Allele 2, respectively.
• a first probe set contains member probes 1001, 1003, 1004.
• 1003 contains a label (1002) of type "A.”
• 1004 contains an affinity tag (1005) which may be used for isolation and identification of the probe product.
• a second probe set with member probes 1001, 1009, 1004 carries respective features as in the first probe set.
• 1001 is identical for both probe sets and 1004 is identical for both probe sets.
• 1009 contains a label (1008) of type "B,” distinguishable from type "A.”
• 1003 and 1009 contain sequences that are nearly identical, and differ by only one nucleotide in the sequence. Therefore, hybridization sequences of these two probes contains complementary regions for Allele 1 (1003), and Allele 2 (1009), respectively. Further, the length of each hybridization domain on 1003 and 1009, as well as experimental hybridization conditions are designed such that probe 1003 will only hybridize to Allele 1 and probe 1009 will only hybridize to Allele 2.
• the purpose of this assay type is to be able to accurately quantify the frequency of Allele 1 and Allele 2 in a sample.
• the probe 1001 contains one or more labels (1000) of type "C.” Therefore, probe products will contain a combination of labels. For Allele 1, probe products will contains labels of type "A” and type "C,” whereas probe products from Allele 2 will contain labels of type "B” and type "C.”
• Figure 31 depicts a modification of the general procedure described in Figure 21 .
• Figure 31 depicts two probe sets for identifying various alleles of the same genomic locus. For example, for distinguishing maternal and fetal alleles, in the case of cell free DNA isolated from a pregnant woman, or for distinguishing host and donor alleles, in the case of cell free DNA from a recipient of an organ transplant.
• Figure 31 depicts two probe sets - one probe set for Allele 1 and one probe set for Allele 2.
• 1104 and 1105 are target molecules corresponding to Allele 1 and Allele 2, respectively.
• a first probe set contains member probes 1101, 1102. 1101 contains a label (1100) of type "A.”
• 1102 contains an affinity tag (1103) which may be used for isolation and identification of the probe product.
• a second probe set with member probes 1107, 1102 carries respective features as in the first probe set. 1102 is identical for both probe sets. However, 1107 contains a label (1106) of type "B," distinguishable from type "A.” 1101 and 1107 contain sequences that are nearly identical, and differ by only one nucleotide in the sequence. Therefore, hybridization sequences of these two probes contains complementary regions for Allele 1 (1101), and Allele 2 (1107). Further, the length of each hybridization domain on 1101 and 1107, as well as experimental hybridization conditions are designed such that probe 1101 will only hybridize to Allele 1 and probe 1107 will only hybridize to Allele 2. The purpose of this assay type is to be able to accurately quantify the frequency of Allele 1 and Allele 2 in a sample.
• the probe 1203 contains one or more labels (1204) of type "C.” Therefore, probe product will contain a combination of labels. For Allele 1, probe products will contains labels of type "A” and type "C,” whereas probe products from Allele 2 will contain labels of type "B” and type "C.”
• Figure 33 depicts a modification of the general procedure described in Figure 21 .
• Figure 33 depicts two probe sets, one probe set for Locus 1 and one probe set for Locus 2, although as aforementioned, multiple probes sets may be designed for each genomic locus.
• 1304 and 1305 are target molecules corresponding to Locus 1 and Locus 2, respectively.
• a first probe sets contains member probes 1301, 1302. 1301 contains a label (1300) of type "A.” 1301 contains an affinity tag (1303) which may be used for isolation and identification of the probe product.
• a second probe set with member probes 1307, 1308 carries respective features as in the first probe set.
• 1307 contains a label (1306) of type "B,” distinguishable from type "A.”
• 1307 contains an affinity tag (1309) which may be identical to or unique from 1303.
• Many probe sets may designed that target "Locus 1,” containing unique probe sequences but the same label type "A.”
• many probe sets may be designed that target "Locus 2,” containing unique probe sequences but the same label type "B.”
• the affinity tags for the many probe sets for Locus 1 may be identical or unique, and the affinity tags for the many probe sets for Locus 2 may be identical or unique.
• the probes 1301 and 1307 have similar structures.
• probe 1301 there are two distinct hybridization domains, such that probe 1302 may be ligated to each end of 1301, forming a probe product consisting of a contiguous, topologically closed molecule of DNA (e.g., a circular molecule).
• the non-hybridizing sequence on probe 1301 may contain additional features, possibly restriction enzyme sites, or primer binding sites for universal amplification.
• probe products are contiguous circular molecules.
• probe products may be isolated from all other nucleic acids via enzymatic degradation of all linear nucleic acid molecules, for example, using an exonuclease.
• 1408 contains a label (1407) of type "B,” distinguishable from type “A.”
• 1408 contains an affinity tag (1411) which may be identical to or unique from 1404.
• Many probe sets may designed that target "Locus 1,” containing unique probe sequences but the same label type "A.”
• many probe sets may be designed that target "Locus 2,” containing unique probe sequences but the same label type "B.”
• the affinity tags for the many probe sets for Locus 1 may be identical or unique, and the affinity tags for the many probe sets for Locus 2 may be identical or unique.
• the probes 1401 and 1408 have similar structures.
• probe 1401 there are two distinct hybridization domains, such that probe 1403 may be ligated to each end of 1401, forming a probe product consisting of a contiguous, topologically closed molecule of DNA (e.g., a circular molecule).
• the non-hybridizing sequence on probe 1401 may contain additional features, possibly restriction enzyme sites, or primer binding sites for universal amplification.
• probe products are contiguous circular molecules.
• probe products may be isolated from all other nucleic acids via enzymatic degradation of all linear nucleic acid molecules, for example, using an exonuclease.
• the probes 1403 and 1410 contain one or more labels (1402, 1409) of type "C.” Therefore, probe products will contain a combination of labels. For Locus 1, probe products will contains labels of type "A” and type "C,” whereas probe products from Locus 2 will contain labels of type "B” and type "C.”
• Figure 35 depicts a modification of the general procedure described in Figure 21 .
• Figure 35 depicts two probe sets, one probe set for Locus 1 and one probe set for Locus 2, although as aforementioned, multiple probes sets may be designed for each genomic locus.
• 1505 and 1506 are target molecules corresponding to Locus 1 and Locus 2, respectively.
• a first probe sets contains member probe 1501.
• 1501 contains a label (1500) of type "A.”
• 1501 contains an affinity tag (1504) which may be used for isolation and identification of the probe product.
• a second probe set with member probe 1508 carries respective features as in the first probe set. However, 1508 contains a label (1507) of type "B,” distinguishable from type "A.”
• 1508 contains an affinity tag (1511) which may be identical to or unique from 1504.
• probe sets may be designed that target "Locus 1,” containing unique probe sequences but the same label type "A.”
• many probe sets may be designed that target "Locus 2,” containing unique probe sequences but the same label type "B.”
• the affinity tags for the many probe sets for Locus 1 may be identical or unique, and the affinity tags for the many probe sets for Locus 2 may be identical or unique.
• the probes 1501 and 1508 have similar structures.
• 1503 may contain one or more labels of type "C,” possibly as a result of incorporation of a nucleotide bearing a label of type "C.”
• This example also conveys to the probe product formed for Locus 2, containing probe 1508.
• the non-hybridizing sequence on probe 1501 and probe 1508 may contain additional features, possibly restriction enzyme sites.
• One feature of this method is that all probe products are contiguous circular molecules. In this manner, probe products may be isolated from all other nucleic acids via enzymatic degradation of all linear nucleic acid molecules, for example, using an exonuclease. Probe products will contain a combination of labels. For Locus 1, probe products will contains labels of type "A” and type "C,” whereas probe products from Locus 2 will contain labels of type "B” and type "C.”
• Figure 36 depicts a modification of the general procedure described in Figure 21 .
• Figure 36 depicts two probe sets, one probe set for Locus 1 and one probe set for Locus 2, although as aforementioned, multiple probes sets may be designed for each genomic locus.
• 1605 and 1606 are target molecules corresponding to Locus 1 and Locus 2, respectively.
• a second probe set with member probe 1609 carries respective features as in the first probe set. However, 1609 contains a label (1608) of type "B,” distinguishable from type "A.” 1609 contains an affinity tag (1607) which may be identical to or unique from 1601. Many probe sets may designed that target "Locus 1,” containing unique probe sequences but the same label type "A.” Similarly, many probe sets may be designed that target "Locus 2,” containing unique probe sequences but the same label type "B.” The affinity tags for the many probe sets for Locus 1 may be identical or unique, and the affinity tags for the many probe sets for Locus 2 may be identical or unique.
• Probes 1602 and 1609 hybridize to sequences corresponding to Locus 1 or Locus 2 respectively, and a DNA polymerase or other enzyme may be used to synthesize a new polynucleotide sequence, for example 1603 in the case of Locus 1 or 1611 in the case of Locus 2.
• 1603 and 1611 may contain one or more labels (1604) of type "C," possibly as a result of incorporation of one of more nucleotides bearing a label of type "C.” This example also conveys to the probe product formed for Locus 2. Therefore, probe products will contain a combination of labels.
• probe products will contains labels of type "A” and type "C,” whereas probe products from Locus 2 will contain labels of type "B” and type “C.” This method results in probe products with high specificity for sequences in Locus 1 or Locus 2 respectively.
• Figure 37 depicts a modification of the general procedure described in Figure 21 .
• Figure 37 depicts two probe sets, one probe set for Locus 1 and one probe set for Locus 2, although as aforementioned, multiple probes sets may be designed for each genomic locus.
• 1704 and 1705 are target molecules corresponding to Locus 1 and Locus 2, respectively.
• a first probe sets contains member probe 1702. 1702 contains an affinity tag (1700) which may be used for isolation and identification of the probe product.
• a second probe set with member probe 1708 carries respective features as in the first probe set.
• 1708 contains an affinity tag (1706) which may be identical to or unique from 1700.
• Many probe sets may designed that target "Locus 1," containing unique probe sequences.
• many probe sets may be designed that target "Locus 2,” containing unique probe sequences.
• the affinity tags for the many probe sets for Locus 1 may be identical or unique, and the affinity tags for the many probe sets for Locus 2 may be identical or unique.
• Probes 1702 and 1708 hybridize to sequences corresponding to Locus 1 and Locus 2 respectively.
• the designs of each probe for Locus 1 and Locus 2 are such that the first adjacent nucleotide next to the hybridization domains contains a different nucleotide for Locus 1 than for Locus 2.
• the first adjacent nucleotide next to the hybridization domain of 1702 is an "A”
• the first adjacent nucleotide next to the hybridization domain of 1708 is a "T”
• All probes for Locus 1 shall be designed such that the first nucleotide immediately adjacent to the hybridization domain shall consist of different nucleotide(s) than the first nucleotide immediately adjacent to the hybridization domain of the probes for Locus 2. That is, by design, probe sets from Locus 1 and Locus 2 may be distinguished from one another based on the identity of the first nucleotide immediately adjacent to the hybridization domain.
• a DNA polymerase or other enzyme will be used to add at least one additional nucleotide to each of the probe sequences.
• the nucleotide substrates for the DNA polymerase are competent for a single addition, for example, the nucleotides may be dideoxy chain terminators. That is, only one new nucleotide shall be added to each probe sequence.
• the nucleotide added to probe 1702 will contain one or more labels (1703) of type "A.”
• the nucleotide added to probe 1708 will contain one or more labels (1709) of type "B," such that the probe products for Locus 1 may be distinguished from the probe products from Locus 2.
• Figure 38 depicts a modification of the general procedure described in Figure 21 .
• Figure 38 depicts two probe sets, one probe set for Locus 1 and one probe set for Locus 2, although as aforementioned, multiple probes sets may be designed for each genomic locus.
• 1804 and 1805 are target molecules corresponding to Locus 1 and Locus 2, respectively.
• a first probe sets contains member probe 1802. 1802 contains an affinity tag (1800) which may be used for isolation and identification of the probe product.
• a second probe set with member probe 1808 carries respective features as in the first probe set.
• 1808 contains an affinity tag (1806) which may be identical to or unique from 1800.
• Many probe sets may be designed that target "Locus 1," containing unique probe sequences.
• many probe sets may be designed that target "Locus 2,” containing unique probe sequences.
• the affinity tags for the many probe sets for Locus 1 may be identical or unique, and the affinity tags for the many probe sets for Locus 2 may be identical or unique.
• Probes 1802 and 1808 hybridize to sequences corresponding to Locus 1 and Locus 2 respectively.
• the designs of each probe for Locus 1 and Locus 2 are such that the first adjacent nucleotide next to the hybridization domains contains a different nucleotide for Locus 1 than for Locus 2.
• the first adjacent nucleotide next to the hybridization domain of 1802 is an "A”
• the first adjacent nucleotide next to the hybridization domain of 1808 is a "T”
• All probes for Locus 1 shall be designed such that the first nucleotide immediately adjacent to the hybridization domain shall consist of different nucleotide(s) than the first nucleotide immediately adjacent to the hybridization domain of the probes for Locus 2. That is, by design, probe sets from Locus 1 and Locus 2 may be distinguished from one another based on the identity of the first nucleotide immediately adjacent to the hybridization domain.
• a DNA polymerase or other enzyme will be used to add at least one additional nucleotide to each of the probe sequences.
• the nucleotide substrates for the DNA polymerase are competent for a single addition, perhaps because the nucleotides added to the reaction mixture are dideoxy nucleotides. That is, only one new nucleotide shall be added to each probe sequence.
• the nucleotide added to probe 1802 will contain one or more labels (1803) of type "A.”
• the nucleotide added to probe 1808 will contain one or more labels (1809) of type "B," such that the probe products for Locus 1 may be distinguished from the probe products from Locus 2.
• probe products 1802 and 1808 contain one or more labels (1801, 1806) of type "C.” Therefore, probe products will contain a combination of labels. For Locus 1, probe products will contains labels of type "A” and type "C,” whereas probe products from Locus 2 will contain labels of type "B” and type "C.”
• Figure 39 depicts a modification of the general procedure described in Figure 21 .
• Figure 39 depicts two probe sets, one probe set for Locus 1 and one probe set for Locus 2, although as aforementioned, multiple probes sets may be designed for each genomic locus.
• 1906 and 1907 are target molecules corresponding to Locus 1 and Locus 2, respectively.
• a first probe set contains member probe 1902.
• 1902 contains an affinity tag (1901) which may be used for isolation and identification of the probe product.
• a second probe set with member probe 1910 carries respective features as in the first probe set.
• 1910 contains an affinity tag (1908) which may be identical to or unique from 1901.
• Many probe sets may be designed that target "Locus 1,” containing unique probe sequences.
• many probe sets may be designed that target "Locus 2,” containing unique probe sequences.
• the affinity tags for the many probe sets for Locus 1 may be identical or unique, and the affinity tags for the many probe sets for Locus 2 may be identical or unique.
• Probes 1902 and 1910 hybridize to sequences corresponding to Locus 1 and Locus 2 respectively.
• the designs of each probe for Locus 1 and Locus 2 are such that the first adjacent nucleotide next to the hybridization domains contains a different nucleotide for Locus 1 than Locus 2.
• the first adjacent nucleotide next to the hybridization domain of 1902 is an "A”
• the first adjacent nucleotide next to the hybridization domain of 1910 is a "T.”
• All probes for Locus 1 shall be designed such that the first nucleotide immediately adjacent to the hybridization domain shall consist of different nucleotide(s) than the first nucleotide immediately adjacent to the hybridization domain of the probes for Locus 2.
• probe sets from Locus 1 and Locus 2 may be distinguished from one another nucleotide on the identity of the first nucleotide immediately adjacent to the hybridization domain.
• a different nucleotide, not one used to distinguish probes from Locus 1 or Locus 2 shall serve as a chain terminator.
• an "A" nucleotide on a target molecule is used do distinguish probes for Locus 1 and a "T" nucleotide is used to distinguish probes for Locus 2.
• a "C" nucleotide may serve as a chain terminator.
• a "C” nucleotide will be added to the assay not is not capable of chain elongation (for example, a dideoxy C).
• the probe sequences are designed such that there are no instances of an identifying nucleotide for Locus 2 present on 1906 in between the distinguishing nucleotide for Locus 1 and the chain terminating nucleotide.
• DNA polymerase or a similar enzyme will be used to synthesize new nucleotide sequences, and the nucleotide added at the distinguishing nucleotide location for Locus 1 will contain one or more labels (1903) of type "A.”
• the nucleotide added at the distinguishing nucleotide location for Locus 2 will contain 1 or more labels (1911) of type "B,” such that the probe products for Locus 1 may be distinguished from the probe products from Locus 2.
• the nucleotide added at the chain terminating position will contain one or more labels (1912) of type "C.” Therefore, probe products will contain a combination of labels. For Locus 1, probe products will contains labels of type "A” and type "C,” whereas probe products from Locus 2 will contain labels of type "B” and type "C.”
• the chain terminator may contain no label.
• a fourth nucleotide may be added to the assay that contains one or more labels of type "C.” This fourth nucleotide does not pair with the identifying nucleotide for Allele 1 (in this example, A), does not pair with the identifying nucleotide for Allele 2 (in this example, T), does not pair with the chain terminating nucleotide (in this example G).
• the fourth nucleotide that would bear one or more labels of type "C” is G, and will pair with C locations on 1906 and 1907. Therefore, probe products will contain a combination of labels. For Locus 1, probe products will contains labels of type "A” and type "C,” whereas probe products from Locus 2 will contain labels of type "B” and type "C.”
• Figure 40 depicts a modification of the general procedure described in Figure 21 .
• Figure 40 depicts two probe sets, one probe set for Locus 1 and one probe set for Locus 2, although as aforementioned, multiple probes sets may be designed for each genomic locus.
• 2005 and 2006 are target molecules corresponding to Locus 1 and Locus 2, respectively.
• a second probe set with member probe 2008 carries respective features as in the first probe set.
• 2008 contains an affinity tag (2007) which may be identical to or unique from 2000.
• Many probe sets may be designed that target "Locus 1," containing unique probe sequences.
• many probe sets may be designed that target "Locus 2,” containing unique probe sequences.
• the affinity tags for the many probe sets for Locus 1 may be identical or unique, and the affinity tags for the many probe sets for Locus 2 may be identical or unique.
• Probes 2001 and 2008 hybridize to sequences corresponding to Locus 1 and Locus 2 respectively.
• the designs of each probe for Locus 1 and Locus 2 are such that there are one or more instances of a distinguishing nucleotide (in this example, "A” is a distinguishing nucleotide for Locus 1 and "T” is a distinguishing nucleotide for Locus 2) followed by a chain terminating nucleotide (in this example "G") adjacent to the hybridization domain of the probes.
• a distinguishing nucleotide for Locus 2 in this example, "T" present in between the hybridization domain of 2001 on 2005 and the chain terminating nucleotide on 2005.
• the distinguishing nucleotide for Locus 1 in this example, "A” present in between the hybridization domain of 2008 on 2006 and the chain terminating nucleotide on 2006.
• DNA polymerase or a similar enzyme will be used to synthesize new nucleotide sequences (2004, 2011) until the addition of a chain terminating nucleotide, one possible example would be a dideoxy C.
• the nucleotides added at the distinguishing nucleotide locations for Locus 1 will contain one or more labels (2003) of type "A.”
• the nucleotides added at the distinguishing nucleotide locations for Locus 2 will contain 1 or more labels (2010) of type "B," such that the probe products for Locus 1 may be clearly distinguished from the probe products from Locus 2.
• Figure 41 depicts a modification of the general procedure described in Figure 21 .
• Figure 41 depicts two probe sets, one probe set for Locus 1 and one probe set for Locus 2, although as aforementioned, multiple probes sets may be designed for each genomic locus.
• 2105 and 2106 are target molecules corresponding to Locus 1 and Locus 2, respectively.
• a first probe sets contains member probe 2102. 2102 contains an affinity tag (2100) which may be used for isolation and identification of the probe product.
• a second probe set with member probe 2109 carries respective features as in the first probe set.
• 2109 contains an affinity tag (2107) which may be identical to or unique from 2100.
• Many probe sets may be designed that target "Locus 1,” containing unique probe sequences.
• many probe sets may be designed that target "Locus 2,” containing unique probe sequences.
• the affinity tags for the many probe sets for Locus 1 may be identical or unique, and the affinity tags for the many probe sets for Locus 2 may be identical or unique.
• Probes 2102 and 2109 hybridize to sequences corresponding to Locus 1 and Locus 2 respectively.
• the designs of each probe for Locus 1 and Locus 2 are such that there are one or more instances of a distinguishing nucleotide (in this example, "A” is a distinguishing nucleotide for Locus 1 and "T” is a distinguishing nucleotide for Locus 2) followed by a chain terminating nucleotide (in this example "G") adjacent to the hybridization domain of the probes.
• a distinguishing nucleotide for Locus 2 in this example, "T” present in between the hybridization domain of 2102 on 2105 and the chain terminating nucleotide on 2105.
• the distinguishing nucleotide for Locus 1 in this example, "A” present in between the hybridization domain of 2109 on 2106 and the chain terminating nucleotide on 2106.
• DNA polymerase or a similar enzyme will be used to synthesize new nucleotide sequences (2104, 2110) until the addition of a chain terminating nucleotide, one possible example would be a dideoxy C.
• the nucleotides added at the distinguishing nucleotide locations for Locus 1 will contain one or more labels (2103) of type "A.”
• the nucleotides added at the distinguishing nucleotide locations for Locus 2 will contain 1 or more labels (2110) of type "B," such that the probe products for Locus 1 may be clearly distinguished from the probe products from Locus 2.
• probe products 2102 and 2109 contain one or more labels (2101, 2108) of type "C.” Therefore, probe products will contain a combination of labels. For Locus 1, probe products will contains labels of type "A” and type "C,” whereas probe products from Locus 2 will contain labels of type "B” and type "C.”
• Figure 42 depicts a modification of the general procedure described in Figure 21 .
• Figure 42 depicts two probe sets for identifying various alleles of the same genomic locus. For example, for distinguishing maternal and fetal alleles, in the case of cell free DNA isolated from a pregnant woman, or for distinguishing host and donor alleles, in the case of cell free DNA from a recipient of an organ transplant.
• Figure 42 depicts two probe sets - one probe set for Allele 1 and one probe set for Allele 2.
• 2203 and 2204 are target molecules corresponding to Allele 1 and Allele 2, respectively.
• a first probe sets contains member probe 2201.
• 2201 contains an affinity tag (2200) which may be used for isolation and identification of the probe product.
• the probe sets used for identification of the two different alleles are the same. That is, the probe set for Allele 2 consists of member probe 2201. Probe 2201 hybridizes to a sequence corresponding to Allele 1 and Allele 2 respectively in Figure 42 .
• the design of probe 2201 is such that the first adjacent nucleotide next to the hybridization domain contains a different nucleotide for Allele 1 than Allele 2.
• the first nucleotide adjacent to the hybridization domain may be a single nucleotide polymorphism, or SNP.
• the first adjacent nucleotide on 2203 next to the hybridization domain of 2201 is an "A”
• the first adjacent nucleotide on 2204 next to the hybridization domain of 2201 is a "T”
• probe products from Allele 1 and Allele 2 may be distinguished from one another based on the identity of the first nucleotide immediately adjacent to the hybridization domain.
• a DNA polymerase or other enzyme will be used to add at least one additional nucleotide to each of the probe sequences.
• the nucleotide substrates for the DNA polymerase are competent for a single addition, perhaps because the nucleotides added to the reaction mixture are dideoxy nucleotides. That is, only one new nucleotide shall be added to each probe sequence.
• the nucleotide added to probe 2201 for Allele 1 will contain one or more labels (2202) of type "A.”
• the nucleotide added to probe 2201 for Allele 2 will contain one or more labels (2205) of type "B,” such that the probe products for Allele 1 may be clearly distinguished from the probe products from Allele 2.
• the probe product for Allele 1 consists of probe 2201 plus one additional nucleotide bearing one or more labels of type "A”
• the probe products for Allele 2 consists of probe 2201 plus one additional nucleotide bearing one or more labels of type "B.”
• Figure 43 depicts a modification of the general procedure described in Figure 21 .
• Figure 43 depicts two probe sets for identifying various alleles of the same genomic locus. For example, for distinguishing maternal and fetal alleles, in the case of cell free DNA isolated from a pregnant woman, or for distinguishing host and donor alleles, in the case of cell free DNA from a recipient of an organ transplant.
• Figure 43 depicts two probe sets - one probe set for Allele 1 and one probe set for Allele 2.
• 2304 and 2305 are target molecules corresponding to Allele 1 and Allele 2, respectively.
• a first probe sets contains member probe 2302. 2302 contains an affinity tag (2300) which may be used for isolation and identification of the probe product.
• the probe sets used for identification of the two different alleles are the same. That is, the probe set for Allele 2 consists of member probe 2302.
• Probe 2302 hybridizes to a sequence corresponding to Allele 1 and Allele 2 respectively in Figure 43 .
• the design of probe 2302 is such that the first adjacent nucleotide next to the hybridization domains contains a different nucleotide for Allele 1 than Allele 2.
• the first nucleotide adjacent to the hybridization domain may be a single nucleotide polymorphism, or SNP.
• the first adjacent nucleotide on 2304 next to the hybridization domain of 2302 is an "A”
• the first adjacent nucleotide on 2305 next to the hybridization domain of 2302 is a "T”
• probe products from Allele 1 and Allele 2 may be distinguished from one another based on the identity of the first nucleotide immediately adjacent to the hybridization domain.
• a DNA polymerase or other enzyme will be used to add at least one additional nucleotide to each of the probe sequences.
• the nucleotide substrates for the DNA polymerase are competent for a single addition, perhaps because the nucleotides added to the reaction mixture are dideoxy nucleotides. That is, only one new nucleotide shall be added to each probe sequence.
• the nucleotide added to probe 2302 for Allele 1 will contain one or more labels (2303) of type "A.”
• the nucleotide added to probe 2302 for Allele 2 will contain one or more labels (2306) of type "B,” such that the probe products for Allele 1 may be clearly distinguished from the probe products from Allele 2.
• the probe product for Allele 1 consists of probe 2302 plus one additional nucleotide bearing one or more labels of type "A”
• the probe products for Allele 2 consists of probe 2302 plus one additional nucleotide bearing one or more labels of type "B.”
• probe products 2302 contain one or more labels (2301) of type "C.” Therefore, probe products will contain a combination of labels. For Allele 1, probe products will contains labels of type "A” and type "C,” whereas probe products from Allele 2 will contain labels of type "B” and type "C.”
• Figure 44 depicts a modification of the general procedure described in Figure 21 .
• Figure 44 depicts two probe sets for identifying various alleles of the same genomic locus. For example, for distinguishing maternal and fetal alleles, in the case of cell free DNA isolated from a pregnant woman, or for distinguishing host and donor alleles, in the case of cell free DNA from a recipient of an organ transplant.
• Figure 44 depicts two probe sets - one probe set for Allele 1 and one probe set for Allele 2.
• 2405 and 2406 are target molecules corresponding to Allele 1 and Allele 2, respectively.
• a first probe sets contains member probe 2401.
• 2401 contains an affinity tag (2400) which may be used for isolation and identification of the probe product.
• the probe sets used for identification of two different alleles are the same. That is, the probe set for Allele 2 consists of member probe 2401.
• Probe 2401 hybridizes to a sequence corresponding to Allele 1 and Allele 2 respectively in Figure 44 .
• the design of probe for 2401 is such that the first adjacent nucleotide next to the hybridization domains contains a different nucleotide for Allele 1 than Allele 2.
• the first nucleotide adjacent to the hybridization domain may be a single nucleotide polymorphism, or SNP.
• the first adjacent nucleotide on 2405 next to the hybridization domain of 2401 is an "A”
• the first adjacent nucleotide on 2406 next to the hybridization domain of 2401 is a "T”
• probe products from Allele 1 and Allele 2 may be distinguished from one another based on the identity of the first nucleotide immediately adjacent to the hybridization domain.
• a DNA polymerase or other enzyme will be used to add at least one additional nucleotide to each of the probe sequences.
• the nucleotide added to probe 2401 for Allele 1 will contain one or more labels (2402) of type "A.”
• the nucleotide added to probe 2401 for Allele 2 will contain one or more labels (2407) of type "B,” such that the probe products for Locus 1 may be clearly distinguished from the probe products from Locus 2.
• the probe product for Allele 1 contains probe 2401 plus an additional nucleotide bearing one or more labels of type "A”
• the probe product for Allele 2 contains probe 2401 plus an additional nucleotide bearing one or more labels of type "B.”
• a different nucleotide, not one used to distinguish Allele 1 from Allele 2 shall serve as a chain terminator.
• an "A” nucleotide on a target molecule is used to identify Allele 1 and a "T" nucleotide is used to identify Allele 2.
• a "C" nucleotide may serve as a chain terminator.
• a "C” nucleotide will be added to the assay that is not is not capable of chain elongation (for example, a dideoxy C).
• the probe sequences are designed such that there are no instances of an identifying nucleotide for Allele 2 is present on 2405 in between the distinguishing nucleotide for Allele 1 an the chain terminating nucleotide.
• DNA polymerase or a similar enzyme will be used to synthesize new nucleotide sequences, and the nucleotide added at the distinguishing nucleotide location for Allele 1 will contain one or more labels (2402) of type "A.”
• the nucleotide added at the distinguishing nucleotide location for Allele 2 will contain 1 or more labels (2407) of type "B,” such that the probe products for Allele 1 may be clearly distinguished from the probe products from Allele 2.
• the nucleotide added at the chain terminating position will contain one or more labels (2403) of type "C.” Therefore, probe products will contain a combination of labels. For Allele 1, probe products will contains labels of type "A” and type "C,” whereas probe products from Allele 2 will contain labels of type "B” and type "C.”
• Figure 45 depicts a modification of the general procedure described in Figure 21 .
• Figure 45 depicts two probe sets for identifying various alleles of the same genomic locus. For example, for distinguishing maternal and fetal alleles, in the case of cell free DNA isolated from a pregnant woman, or for distinguishing host and donor alleles, in the case of cell free DNA from a recipient of an organ transplant.
• Figure 45 depicts two probe sets - one probe set for Allele 1 and one probe set for Allele 2.
• 2505 and 2506 are target molecules corresponding to Allele 1 and Allele 2, respectively.
• a first probe sets contains member probe 2501. 2501 contains an affinity tag (2500) which may be used for isolation and identification of the probe product.
• the probe sets used for identification of two different alleles are the same. That is, the probe set for Allele 2 consists of member probe 2501.
• Probe 2501 hybridizes to a sequence corresponding to Allele 1 and Allele 2 respectively in Figure 45 .
• the design of probe for 2501 is such that the first adjacent nucleotide next to the hybridization domains contains a different nucleotide for Allele 1 than Allele 2.
• the first nucleotide adjacent to the hybridization domain may be a single nucleotide polymorphism, or SNP.
• the first adjacent nucleotide on 2505 next to the hybridization domain of 2501 is an "A”
• the first adjacent nucleotide on 2506 next to the hybridization domain of 2501 is a "T”
• probe products from Allele 1 and Allele 2 may be distinguished from one another based on the identity of the first base immediately adjacent to the hybridization domain.
• a DNA polymerase or other enzyme will be used to add at least one additional nucleotide to each of the probe sequences.
• the nucleotide added to probe 2501 for Allele 1 will contain one or more labels (2502) of type "A.”
• the nucleotide added to probe 2501 for Allele 2 will contain one or more labels (2507) of type "B,” such that the probe products for Locus 1 may be clearly distinguished from the probe products from Locus 2.
• the probe product for Allele 1 contains probe 2501 plus an additional nucleotide bearing one or more labels of type "A”
• the probe product for Allele 2 contains probe 2501 plus an additional nucleotide bearing one or more labels of type "B.”
• a different nucleotide, not one used to distinguish Allele 1 from Allele 2 shall serve as a chain terminator.
• an "A” nucleotide on a target molecule is used to identify Allele 1 and a "T" nucleotide is used to identify Allele 2.
• a "C" nucleotide may serve as a chain terminator.
• a "C” nucleotide will be added to the assay that is not is not capable of chain elongation (for example, a dideoxy C).
• the probe sequences are designed such that no instances of an identifying nucleotide for Allele 2 are present on 2505 in between the distinguishing nucleotide for Allele 1 and the chain terminating nucleotide.
• a fourth nucleotide may be added to the assay that contains one or more labels (2508, 2503) of type "C.”
• This fourth nucleotide does not pair with the identifying nucleotide for Allele 1 (in this example, A), does not pair with the identifying nucleotide for Allele 2 (in this example, T), does not pair with the chain terminating nucleotide (in this example G).
• the fourth nucleotide that would bear one or more labels of type "C” is G, and will pair with C locations on 2505 and 2506. Therefore, probe products will contain a combination of labels. For Allele 1, probe products will contains labels of type "A” and type "C,” whereas probe products from Allele 2 will contain labels of type "B” and type "C.”
• Figure 46 depicts a modification of the general procedure described in Figure 21 .
• Figure 46 depicts two probe sets for identifying various alleles of the same genomic locus. For example, for distinguishing maternal and fetal alleles, in the case of cell free DNA isolated from a pregnant woman, or for distinguishing host and donor alleles, in the case of cell free DNA from a recipient of an organ transplant.
• Figure 46 depicts two probe sets - one probe set for Allele 1 and one probe set for Allele 2.
• 2605 and 2606 are target molecules corresponding to Allele 1 and Allele 2, respectively.
• a first probe set contains member probe 2602.
• 2602 contains a label (2601) of type "A.”
• 2602 contains an affinity tag (2600) which may be used for isolation and identification of the probe product.
• 2602 and 2609 contain sequences that are nearly identical, and differ by only one nucleotide in the sequence. Therefore, hybridization sequences of these two probes are complementary to Allele 1 (2605), or Allele 2 (2606). Further, the length of each hybridization domain on 2602 and 2609, as well as experimental hybridization conditions are designed such that probe 2602 will only hybridize to Allele 1 and probe 2609 will only hybridize to Allele 2. The purpose of this assay type is to be able to accurately quantify the frequency of Allele 1 and Allele 2 in a sample.
• the right arm of the Locus 2 probe set consists of a homolog to Locus 2 sequence and a reverse priming sequence for labeling the Locus 2 probe set with label B.
• the forward priming sequence and the affinity tag sequence are identical for the probe sets for both Locus 1 and Locus 2.
• the homologous sequences are specific to a single genomic locus. Locus homologous sequences for each probe set are immediately adjacent to one another such that when they hybridize to their target loci, they immediately abut one another and thus may be ligated to form one continuous molecule.
• the reverse priming sequence is specific to the label (e.g., label A or label B) to be used in labeling probe products for a particular locus for a particular affinity tag sequence.
• Figure 56 depicts the procedural workflow that would be applied to the collection of probe sets, such as those probe sets illustrated in Figure 55 .
• This depiction is based on one probe set for one genomic locus (e.g., the probe set for Locus 1 shown in Figure 55 ).
• the collection of probe sets is mixed with purified cell-free DNA.
• the locus specific sequences in each probe set hybridize to their corresponding homologous sequences in the cell-free DNA sample.
• a ligase enzyme is added to catalyze the formation of a phosphodiester bond between the 3' base on the left arm homolog and the 5' arm of the right homolog, closing the nick between the two arms and thus forming one continuous molecule which is the probe product.
• Figure 57 depicts a modified version of the procedural workflow illustrated in Figure 56 .
• the left arm of each probe set contains a terminal biotin molecule as indicated by a "B" in Steps 1 to 6 of the Figure.
• This biotinylation enables the purification of the collection of probe products after completion of the hybridization-ligation reaction and prior to the PCR amplification.
• the workflow for this method is identical to that described in Figure 57 for Steps 1 to 3.
• Step 4 streptavidin-coated magnetic beads are added to the hybridization-ligation reaction.
• the biotin molecule contained in the probe products will bind the products to the streptavidin.
• Figure 59 provides evidence that probe products representing a multitude of genomic locations for one locus may be generated in a ligase enzyme specific manner using the hybridization-ligation process.
• Eight probe sets each consisting of a left arm and right arm component as described in Figure 55 and, containing homologs to eight chromosome 18 locations were hybridized to synthetic oligonucleotide templates (about 48 nucleotides) and ligated using a ligase enzyme to join the left and right arms for each. Reaction products were analyzed using denaturing polyacrylamide gel electrophoresis. Gel lane 1 contains a molecular weight ladder to indicate DNA band sizes. Lanes 2 to 9 contain hybridization-ligation reaction products for the eight chromosome 18 probe sets.
• Quantitative PCR was used to determine the amount of probe product present for each cell line following the hybridization-ligation and purification processes described in Figure 57 (Steps 1 to 5).
• the copy number state measured for the various cell lines followed the expected trend indicated in Table 1.
• qPCR indicated a copy number state of less than two for NA12138, which has one copy of chromosome X.
• the measured copy number state for NA00254 (three copies of X) was greater than two, for NA01416 (four copies of X) was greater than three, and for NA06061 (five copies of X) was greater than four.
• the responsiveness of the process in detecting differences in copy number state is further illustrated by Figure 60B in which the measured copy number state is plotted against the theoretical copy number state.
• Figure 61A depicts representative fluorescence images of two array spots in two orthogonal imaging channels (Alexa 488: green, Alexa 594; red).
• a region of interest (ROI) is automatically selected (large circle), with any undesired bright contaminants being masked from the image (smaller outlined regions within the ROI).
• Single fluorophores on single hybridized assay products are visualized as small punctate features within the array spot.
• a "Balanced" spot (representing genomic targets input at a 1:1 concentration ratio to the assay) imaged in the green channel and (ii) the same spot imaged in the red channel.
• An "Increased" spot (representing genomic targets input at a > 1:1 concentration ratio to the assay) imaged in the green channel and (iv) the same spot imaged in the red channel.
• Figure 61B presents raw counts of the detected fluorophores in two channels for five spots each of the "Balanced” and “Increased” conditions. Despite some variation in the absolute number of fluors, the numbers in the two channels track closely for the "Balanced” case, but demonstrate clear separation in the "Increased” case.
• Figure 61C presents calculated ratio values for number of fluors in the green channel divided by the number of fluors in the red channel, for the five spots from each of the "Balanced” and “Increased” conditions.
• the "Balanced” case centers about a ratio of 1.0 and the “Increased” case is at an elevated ratio.
• the "Balanced” case as comparing two balanced genomic loci and the "Increased” case as one where one locus is increased relative to the other, we may calculate the confidence of separation of the two conditions using an independent, 2-group T-test, yielding a p-value of 8 x 10 -14 .
• Figure 62 illustrates a modification of the general procedure described in Figures 55 to 58 .
• a second probe set, Probe Set B is designed for each genomic location such that the genome homolog sequences in Probe Set B are a reverse complement of the genome homolog sequences in Probe Set A.
• Probe Set A will hybridize to the reverse strand of the genomic DNA and Probe Set B will hybridize to the forward strand of the genomic DNA. This will provide increased sensitivity relative to the embodiment described in Figures 55 to 58 as it will yield approximately double the number of probe products per locus.
• Figure 63 illustrates a modification to the general procedure described in Figure 57 .
• the Reverse Primer used in Step 6 is additionally modified in that the four bonds linking the first five nucleotides in the oligonucleotide sequence are phosphorothioate bonds.
• This modification will result in all PCR products generated during PCR amplification (Step 7) having a phosphorothioate modification on the 5' end. This modification will protect the reverse strand from any digestion that might occur during the treatment with Lambda exonuclease in Step 8.
• Figure 64 illustrates a modification of the general procedure described in Figures 55 to 58 .
• PCR amplification of the probe product is replaced with linear amplification by adding the Reverse Primer but no Forward Primer to the amplification reaction in Step 6. If only the Reverse Primer is present the amplification product will be single stranded - the reverse strand with a label of the 5' end.
• the amplification product is already single-stranded, it does not require further processing before hybridization to a microarray, i.e., Lambda exonuclease digestion may be omitted.
• a forward primer is not used in this embodiment, it is unnecessary for the left arm of the probe set to contain a forward priming sequence. The left arm would consist of an affinity tag sequence and a locus homolog sequence only as illustrated in Figure 64 .
• a further method of the general procedure described in Figures 55 to 58 is one in which the single ligation reaction process in Step 3 is replaced with a cycled ligation reaction process. This is accomplished by replacing the thermolabile ligase enzyme (e.g., T4 ligase) used to catalyze the ligation reaction with a thermostable ligase (e.g., Taq ligase).
• a thermostable ligase e.g., Taq ligase
• the hybridization-ligation reaction may be heated to a temperature that will melt all DNA duplexes (e.g., 95 °C) after the initial cycle of hybridization and ligation has occurred. This will make the genomic template DNA fully available for another probe set hybridization and ligation.
• thermocycling of the hybridization and ligation reaction between a temperature that will melt DNA duplexes and one that will allow hybridization and ligation to occur will linearly increase the amount of probe product yielded from the reaction. If the reaction is exposed to 30 such cycles, up to 30 times the amount of probe product will be yielded than from a process in which a single ligation reaction is used.
• Figures 65A and 65B depict further methods of the modified procedure described in Figure 62 .
• This method takes advantage of the ligase chain reaction (LCR) in combining the presence of the reverse complement for each probe set with the use of a thermostable ligase to enable a cycled ligation reaction in which the product is exponentially amplified.
• Figures 65A and 65B depict two probe sets, Probe Set A and Probe Set B for one locus; where the genome homolog sequences in Probe Set B are the reverse complement of the genome homolog sequences in Probe Set A.
• the 5' arm of each Probe Set consists of an affinity tag sequence and a homolog while the 3' arm of each Probe Set consists of a homolog sequence with a label attached.
• Figures 66A, 66B, and 66C depict yet other methods of the procedure depicted in Figures 65A and 65B .
• the 5' arm of each Probe Set consists of an affinity tag sequence and a homolog while the 3' arm of each Probe Set consists of a homolog sequence and a priming sequence without a label attached as illustrated in Figure 66A .
• the probe product may be purified.
• the LCR product would then be amplified in a linear manner by the addition of a single primer that has a label attached, along with reaction components ( Taq polymerase, dNTPs, and reaction buffer) as illustrated in Figure 66B .
• the product of this amplification would be single-stranded (reverse strand only) with a 5' label as illustrated in Figure 66C . Consequently it would not be necessary to treat it with Lambda exonuclease but rather it could instead be directly used as microarray target.
• a significant challenge in oncology is the early detection of cancer. This is particularly true in cancers that are hard to image or biopsy (e.g., pancreatic cancer, lung cancer).
• Cell free tumor DNA (tumor cfDNA) in a patient's blood offers a method to non-invasively detect a tumor. These may be solid tumors, benign tumors, micro tumors, liquid tumors, metastasis or other somatic growths. Detection may be at any stage in the tumor development, though ideally early (Stage I or Stage II). Early detection allows intervention (e.g., surgery, chemotherapy, pharmaceutical treatment) that may extend life or lead to remission.
• probe sets may be configured to target known genetic variations associated with tumors. These may include mutations, SNPs, copy number variants (e.g., amplifications, deletions), copy neutral variants (e.g., inversions, translocations), and/or complex combinations of these variants.
• known genetic variations associated with tumors include those listed in cancer.sanger.ac.uk/cancergenome/projects/cosmic;
• inversions that occur at known locations may easily be targeted by designing probes that at least partially overlap the breakpoint in one probe arm.
• a first probe that binds the "normal" sequence targets non-inverted genomic material ( Figure 67B ) and carries a first label type.
• a second probe that binds the "inverted” target carries a second label type ( Figure 67C ).
• a common right probe arm binds native sequence that is not susceptible to inversion, immediately adjacent the first two probes. This right probe arm further carries a common pull-down tag that localizes the probe products to the same region of an imaging substrate. In this way, the probe pairs may hybridize to the genomic targets, ligate, and be imaged to yield relative counts of the two underlying species.
• translocations that have known breakpoints may also be assayed.
• Figure 68A shows two genetic elements that are either in their native order or translocated. Probe arms that at least partially overlap these translocation breakpoints allow differentiation between normal and transposed orders of genetic material. As shown in Figures 68B and 68C , by choosing unique labels on the two left arms, the resulting ligated probe products may be distinguished and counted during imaging.
• copy neutral changes e.g., inversions, translocation
• methods for detecting copy neutral changes may also be used to detect germline variants in cancer or in other disease or conditions.
• left probe arms are designed to take advantage of an energetic imbalance caused by one or more mismatched SNPs. This causes one probe arm (1101, carrying one label) to bind more favorably than a second probe arm (1107, carrying a second type of label). Both designs ligate to the same right probe arm (1102) that carries the universal pull-down tag.
• a given patient's blood may be probed by one method, or a hybrid of more than one method. Further, in some cases, customizing specific probes for a patient may be valuable. This would involve characterizing tumor features (SNPs, translocations, inversions, etc.) in a sample from the primary tumor (e.g., a biopsy) and creating one or more custom probe sets that is optimized to detect those patient-specific genetic variations in the patient's blood, providing a low-cost, non-invasive method for monitoring. This could have significant value in the case of relapse, where detecting low-level recurrence of a tumor type (identical or related to the original tumor) as early as possible is ideal.
• SNPs tumor features
• translocations e.g., translocations, inversions, etc.
• probes may be designed to monitor current status and progression "checkpoints," and guide therapy options.
• the ALK translocation has been associated with lung cancer.
• a probe designed to interrogate the ALK translocation may be used to detect tumors of this type via a blood sample. This would be highly advantageous, as the standard method for detecting lung tumors is via a chest x-ray an expensive procedure that may be deleterious to the patient's health and so is not standardly performed.
• Detection of non-primary tumor types For example, a HER2+ breast tumor is removed by surgery and the patient is in remission. A probe targeting the EGFR gene may be used to monitor for EGFR+ tumors. If these are detected, the patient may have a second EGFR+ tumor either at the primary site or elsewhere.
• Detection of metastasis For example, the patient has a HER2+ breast tumor.
• a probe designed to interrogate the ALK translocation may be used to detect tumors of this type via a blood sample. This tumor may not be in the breast and is more likely to be in the lung. If these are detected, the patient may have a metastatic tumor distal to the primary organ.
• tumor heterogeneity Many tumors have multiple clonal populations characterized by different genetic variants. For example, a breast tumor may have one population of cells that are HER2+ and another population of cells that are EGFR+. Using probes designed to target both these variants would allow the identification of this underlying genetic heterogeneity.
• the quantity of tumor cfDNA may be measured and may be used to determine the size, growth rate, aggressiveness, stage, prognosis, diagnosis and other attributes of the tumor and the patient. Ideally, measurements are made at more than one time point to show changes in the quantity of tumor cfDNA.
• a HER2+ breast tumor is treated with Herceptin.
• a probe targeting the HER2 gene may be used to monitor for quantity of tumor cfDNA, which may be a proxy for the size of the tumor. This may be used to determine if the tumor is changing in size and treatment may be modified to optimize the patient's outcome. This may include changing the dose, stopping treatment, changing to another therapy, combing multiple therapies.
• the present invention offers a way to detect tumors at some or all locations in the body.
• a panel of probes is developed at a spacing of 100 kb across the genome. This panel may be used as a way to detect genetic variation across the genome.
• the panel detects copy number changes of a certain size across the genome. Such copy number changes are associated with tumor cells and so the test detects the presence of tumor cells.
• Different tumor types may produce different quantities of tumor cfDNA or may have variation in different parts of the genome. As such, the test may be able to identify which organ is affected. Further the quantity of tumor cfDNA measured may indicate the stage or size of the tumor or the location of the tumor. In this way, the test is a whole-genome screen for many or all tumor types.
• a threshold may be used to determine the presence or certainty of a tumor. Further, the test may be repeated on multiple samples or at multiple time points to increase the certainty of the results. The results may also be combined with other information or symptoms to provide more information or more certain information on the tumor.
• Exemplary probe sets and primers that may be used in the method described herein to measure copy number of nucleic acid regions of interest are listed in Table 4 below.
• Each of the exemplary probe sets in Table 4 comprises two probes.
• the first (tagging) probe has a structure including a forward priming site, tag, and homology 1.
• the second (labeling) probe has structure, including homology 2 and reverse primer site, which is used in labeling.
• the component sequences of the probes (tag, homology sequence etc.) are also shown.
• Table 4 Exemplary probes and primers.
• Exemplary probe sets and primers that may be used in the method described herein to detect a polymorphism at a SNP site are listed in Table 5 below.
• Each of the exemplary probe sets in Table 5 comprises three probes, two allele specific probes (that are used for labeling) and a tagging probe.
• the two allele specific probes have homology sequences that are different at one or more nucleotides.
• the structure of the first allelic probe includes a Forward Primer Site Allele 1 and Homology Allele 1; and the structure of the second allelic probe includes a Forward Primer Site Allele 2 and Homology Allele 2.
• labeled primers may be used with different labels on the two primers (so the labels are allele specific).
• a universal 3' probe which includes a homology region (without any SNP), the tagging sequence and a reverse primer site.
• the component sequences of the probes (tag, homology sequence etc.) are also shown.
• Table 5 Exemplary probes and primers.
• Cell-free DNA in a volume of 20 ⁇ L water
• Probe Mix mixture of all Tagging and Labeling probe oligonucleotides at a concentration of 2 nM each
• Taq Ligase 40 U/ ⁇ L
• Magnetic Beads MyOne Streptavidin C1 Dynabeads
• Bead Binding and Washing Buffer 1X and 2X concentrations
• Forward amplification primer 5' phosphate modified
• Reverse amplification primer labeled
• AmpliTaq Gold Enzyme (5 U/ ⁇ L)
• dNTP Mix Lambda Exonuclease (5 U/ ⁇ L)
• Hybridization Buffer 1.25X
• Hybridization control oligonucleotides Microarray Wash Buffer A; Microarray Wash Buffer B; Microarray Wash Buffer C
• Hybridization-ligation Reaction The cfDNA samples (20 ⁇ L) were added to wells A3-H3 of a 96-well reaction plate. The following reagents were added to each cfDNA sample for a total reaction volume of 50 ⁇ L, and mixed by pipetting up and down 5-8 times. Component Volume H 2 O 19.33 ⁇ L Probe Mix 5 ⁇ L 10X Taq Ligase Buffer 5 ⁇ L Taq Ligase 0.67 ⁇ L
• the plate was placed in a thermal cycler and ligate using the following cycling profile: (i) 95 °C for 5 minutes; (ii) 95 °C for 30 seconds; (iii) 45 °C for 25 minutes; (iv) Repeat steps b to c 4 times; and (v) 4 °C hold.
• Wash Dynabeads a vial of Dynabeads was vortexted at highest setting for 30 seconds. 260 ⁇ L beads were transferred to a 1.5 mL tube. 900 ⁇ L of 2X Bead Binding and Washing Buffer and mix beads were mixed by pipetting up and down 5-8 times. The tube was placed on a magnetic stand for 1 min, and the supernatant was discarded. The tube from the magnetic stand was removed and resuspended the washed magnetic beads in 900 ⁇ L of 2X Bead Binding and Washing Buffer by pipetting up and down 5-8 times. The tube was placed on the magnetic stand for 1 min and discard the supernatant. The tube was removed from the magnetic stand and add 1,230 ⁇ L of 2X Bead Binding and Washing Buffer. The beads were resuspended by pipetting up and down 5-8 times.
• Immobilize HL Products 50 ⁇ L of washed beads was transferred to each hybridization-ligation reaction product in the 96-well reaction plate and mix by pipetting up and down 8 times, was incubated for 15 min at room temperature, mixed on a plate magnet twice during the incubation time. The beads were separated with on a plate magnet for 3 min and then remove and discard the supernatant. The plate was removed from the plate magnet, 200 ⁇ L 1X Bead Binding and Washing Buffer were added, and the beads were resuspended by pipetting up and down 5-8 times. The plate was placed on the plate magnet for 1 min, and the supernatant was discarded. The plate was removed from the plate magnet, 180 ⁇ L 1X SSC was added, and the beads were resuspended by pipetting up and down 5-8 times. The plate was placed on the plate magnet for 1 min, and the supernatant was discarded.
• the plate was removed from the plate magnet, and 180 ⁇ L 0.1 M NaOH was added, and the beads were resuspended by pipetting up and down 5-8 times. The plate was placed on the plate magnet for 1 min, and the supernatant was discarded. The plate was removed from the plate magnet, 200 ⁇ L of 1X Binding and Wash Buffer were added, and the beads were resuspended by pipetting up and down 5-8 times. Place the plate on the plate magnet for 1 min and discard the supernatant. Remove the plate from the plate magnet, add 180 ⁇ L TE, and the beads were resuspended by pipetting up and down 5-8 times. The plate was placed on the plate magnet for 1 min, and the supernatant was discarded. 20 ⁇ L water was added to each well and the beads were resuspended by pipetting up and down 5-8 times. The plate was sealed and store at 4 °C until used in subsequent steps.
• the plate was placed in a thermal cycler, and the probes were ligated using the following cycling profile: (i) 95 °C for 5 minutes; (ii) 95 °C for 30 seconds; (iii) 45 °C for 25 minutes; (iv) Repeat steps b to c 4 times; and (v) 4 °C hold.
• Hybridization-ligation Product Purification the reagents were mixed by pipetting up and down 5-8 times. The plate was placed in a thermal cycler, and the probes were amplified using the following cycling profile: (i) 95 °C for 5 minutes; (ii) 95 °C for 30 seconds; (iii) 54 °C for 30 seconds; (iv) 72 °C for 60 seconds, (v) Repeat steps b to d 29 times; (vi) 72 °C for 5 minutes; (vii) Repeat steps b to c 4 times; and (v) 4 °C hold.
• Microarray Target Preparation single strand digestion: the following reagents were added to each amplified reaction product in the 96-well reaction plate for a total reaction volume of 60 ⁇ L.
• the reagents were mixed by pipetting up and down 5-8 times.
• the plate was placed in a thermal cycler, and the probes were digested using the following cycling profile: (i) 37 °C for 60 minutes; (ii) 80 °C for 30 minutes; (iii) 4 °C hold.
• the plate was placed in Speed-vac and dry down samples using medium heat setting for about 60 minutes or until all liquid has evaporated. Samples were stored at 4 °C in the dark until used in subsequent steps.
• Microarray Target For each sample, 15 ⁇ L of Microarray Target was added to the center of a Lifter Slip in a hybridization chamber, and the appropriate microarray was immediately placed onto the target fluid by placing the top edge down onto the lifter slip and slowly letting it fall down flat.
• the hybridization chambers were closed and incubated them at 42 °C for 60 minutes.
• the hybridization chambers were opened, and each microarray was removed from the Lifter Slips and placed into a rack immersed in Microarray Wash Buffer A. Once all the microarrays were in the rack, the rack was stirred at 650 rpm for 5 minutes.
• the rack of microarrays was removed from Microarray Wash Buffer A, excess liquid on a clean room wipe was tapped off , and the rack were quickly placed into Microarray Wash Buffer B. The rack was stirred at 650 rpm for 5 minutes. The rack of microarrays was removed from Microarray Wash Buffer B, excess liquid was tapped off on a clean room wipe, and the rack was quickly placed into Microarray Wash Buffer C. The rack was stirred at 650 rpm for 5 minutes. Immediately upon completion of the 5 minute wash in Microarray Wash Buffer C, the rack of microarrays was slowly removed from the buffer. This took 5-10 seconds to maximize the sheeting of the wash buffer from the cover slip surface. Excess liquid was tapped off on a clean room wipe. A vacuum aspirator was used to remove any remaining buffer droplets present on either surface of each microarray. The microarrays were stored in a slide rack under nitrogen and in the dark until the microarrays were analyzed.

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